Mammalian TNF-alpha convertases

ABSTRACT

The present invention provides isolated human and bovine TNF-α convertases, nucleic acids and recombinant vectors encoding the same, host cells comprising the nucleic acids and vectors, and methods for making the convertases using the host cells. This invention further provides antibodies and antigen binding fragments thereof which specifically bind to the convertases and are useful for treating medical conditions caused or mediated by TNF-α. Also provided are screening methods for identifying specific inhibitors of mammalian TNF-α convertases, and for identifying nucleic acids encoding such convertases.

[0001] This application claims the benefit of U.S. ProvisionalApplication 60/021,710, filed Jul. 12, 1996.

TECHNICAL FIELD

[0002] The present invention relates to mammalian tumor necrosisfactor-α (TNF-α) convertase enzymes. More particularly, it relates tobovine, human and other TNF-α convertases, isolated nucleic acids andrecombinant vectors encoding the enzymes, methods for making theenzymes, fragments or fusion proteins thereof using recombinant DNAmethodology or chemical synthesis, and to methods for using the enzymesin screening systems to identify TNF-α convertase inhibitors for thetreatment of various diseases, and nucleic acids encoding a TNF-αconvertase. This invention further relates to antibodies, bothpolyclonal and monoclonal, which specifically bind to the TNF-αconvertases, and to fragments and fusion proteins of the TNF-αconvertases of the invention.

BACKGROUND OF THE INVENTION

[0003] TNF-α, also known as cachectin, is a 17 kDa (kilodalton) proteinproduced by cells of the monocyte/macrophage lineage, and by othercells. A variety of biological effects, both beneficial and deleterious,have been attributed to TNF-α. TNF-α is beneficial, e.g., in that it isbelieved to be a part of host anti-tumor defenses. It also producesdetrimental effects, however, including, e.g., cardiovascular (shock,ARDS, capillary leakage syndrome), renal (nephritis, acute tubalnecrosis), and gastrointestinal (ischemia, colitis, hepatic necrosis)effects, and effects on the central nervous system (fever, anorexia,altered pituitary hormone secretion). In view of the foregoing, aconsensus view has developed that TNF-α is a key mediator ofinflammation (including inflammatory diseases such as arthritis) andmammalian responses to injury, invasion by pathogens, and neoplasia.

[0004] The biosynthesis of human TNF-α proceeds by way of amembrane-bound precursor containing 233 amino acid residues [Wang etal., Science 228:149-154 (1985); Muller et al., Nature 335:265-267(1987)], which is processed during cellular activation by cleavage of a76-residue peptide to produce the mature, secreted form of TNF-α. Theenzyme(s) responsible for this cleavage, called TNF-α convertase, hasuntil the present invention been elusive for most mammalian species.

[0005] A putative TNF-α convertase, called PR-3, has been isolated andcloned from human neutrophils, and it has been suggested that thisenzyme can be used in screens to identify TNF-α convertase inhibitors.See International Patent Applications Publication Numbers WO 94/00555and WO 95/24501. This enzyme, however, is not believed to be thephysiologically relevant human TNF-α convertase because it is a serineprotease, whereas the relevant enzyme is believed to be ametalloproteinase. Moreover, the source of the serine protease,neutrophils, is not believed to be important in the production of TNF-α,and the serine protease does not cleave the precursor form of TNF-α(proTNF-α) at the point expected for the physiologically relevant humanenzyme.

[0006] Mohler et al. [Nature 70:218 (1994)] have partially purifiedanother TNF-α convertase from the human monocytic cell line THP-1. Thispreparation, however, was very impure, and little could be said aboutthe nature of the TNF-α convertase in the crude protein mixture ofMohler et al.

[0007] In view of the important role of TNF-α in many disease processes,there is a need for agents that can selectively block the biosynthesisof mature, secreted TNF-α. The search for such agents would be greatlyfacilitated by the availability of substantially pure mammalian TNF-αconvertases.

SUMMARY OF THE INVENTION

[0008] The present invention fills the foregoing need by providingmaterials and methods for identifying specific inhibitors of TNF-αconvertase. More particularly, this invention provides substantiallypure mammalian TNF-α convertases capable of converting proTNF-α to themature, secreted form. This invention further provides isolated orrecombinant nucleic acids encoding mammalian TNF-α convertases, andrecombinant vectors and host cells comprising such nucleic acids.

[0009] This invention further provides a method for making a mammalianTNF-α convertase, comprising culturing a host cell comprising a nucleicacid encoding a mammalian TNF-α convertase under conditions in which thenucleic acid is expressed. In some embodiments, the method furthercomprises isolation of the TNF-α convertase from the culture.

[0010] This invention also provides polypeptides comprising a fragmentof a TNF-α convertase having an amino acid sequence corresponding to thesequence of at least about 8 contiguous residues of the complete enzymesequence. Preferably, the polypeptides comprise at least about 12, morepreferably at least about 20, and most preferably at least about 30 suchresidues.

[0011] Still further, this invention provides fusion proteins comprisinga TNF-α convertase or a polypeptide thereof covalently linked to afusion partner.

[0012] The present invention also provides antibodies, both polyclonaland monoclonal, that specifically bind to one or more of the TNF-αconvertases or to a polypeptide thereof. Also provided areanti-idiotypic antibodies, both monoclonal and polyclonal, whichspecifically bind to the foregoing antibodies. This invention stillfurther provides a method of treatment comprising administering to amammal afflicted with a medical condition caused or mediated by TNF-α,an effective amount of an antibody, or an antigen-binding fragmentthereof, that specifically binds to a mammalian TNF-α convertase, andpharmaceutical compositions comprising such antibodies or fragments andpharmaceutically acceptable carriers.

[0013] The present invention also provides a method for identifying aninhibitor of a mammalian TNF-α convertase, comprising:

[0014] (a) contacting a mammalian TNF-α convertase in the presence ofsubstrate with a sample to be tested for the presence of an inhibitor ofthe convertase; and

[0015] (b) measuring the rate of cleavage of the substrate;

[0016] whereby an inhibitor of the TNF-α convertase in the sample isidentified by measuring substantially reduced cleavage of the substrate,compared to what would be measured in the absence of such inhibitor.

[0017] In a preferred embodiment, the contacting of the convertase withthe sample in the presence of substrate occurs on the surface of amammalian host cell comprising one or more nucleic acids encoding amammalian TNF-α convertase and a substrate of the convertase.

BRIEF DESCRIPTION OF THE FIGURES

[0018] The present invention can be more readily understood by referenceto the following Description and Examples, and to the accompanyingFigures, in which:

[0019]FIG. 1 is an elution profile from an HPLC column showingDNP-proTNF-α cleavage products;

[0020]FIG. 2 is a graphical representation of results from an assay inwhich human proTNF-α was cleaved by membrane-type matrixmetalloproteases MT-MMP1 and MT-MMP3; and

[0021]FIG. 3 is a graphical representation of results from an assay inwhich cleavage of human proTNF-α by MT-MMP1 was inhibited by varyingamounts of an MMP inhibitor.

DESCRIPTION OF THE INVENTION

[0022] All references cited herein are hereby incorporated in theirentirety by reference. As used herein, the terms “proteinase” and“protease” are intended to mean the same thing and are usedinterchangeably. So too are the terms “assay(s)” and “screen(s)”.

[0023] Characterization of TNF-α Convertases

[0024] The mammalian TNF-α convertases of the present invention arefunctionally characterized by an ability to process, through proteolyticcleavage, the conversion of the membrane-bound form of a precursor formof TNF-α, referred to herein as “proTNF-α”, to the soluble, mature form.This processing entails cleavage of the first 76 amino-terminal residuesof the human precursor protein, the entire sequence of which is definedin the Sequence Listing by SEQ ID NO: 1. This sequence, taken from HumanCytokines, B. Aggarwal and J. Gutterman, Eds., 1992, BlackwellScientific Publications, Oxford, pp. 276-277, is in agreement with theSwiss-Prot sequence, Accession Code: Swiss-Prot P01375. There is aconflict, however, with the corresponding GenBank sequence [AccessionNo. M10988; Wang et al., Science 228:149 (1985)], which has a serineresidue at position −14, instead of the phenylalanine residue shown atthat position in SEQ ID NO: 1.

[0025] Regardless of whether one or both of these sequences is correct,as might be the case with allelic or polymorphic variants, cleavage ofproTNF-α by the TNF-α convertases of this invention occurs at an Ala-Valpeptide bond, resulting in mature human TNF-α having a valine residue atthe amino terminus (i.e., beginning with the Val residue at position 1of SEQ ID NO: 1).

[0026] The foregoing cleavage point is different from that observed forthe serine protease PR-3 mentioned above. Robache-Gallea et al. [J.Biol. Chem. 270:23688 (1995)] have shown that the serine proteasecleaves between Val₁ and Arg² of SEQ ID NO: 1, thereby producing amature form of TNF-α having an N-terminal arginine residue.

[0027] The mammalian TNF-α convertases of the present invention arefurther characterized by their presence in cells that make TNF-α. Theymay be present in other cells as well, however, and may even beubiquitously expressed. Control could be exerted at the level oftranscription of the proTNF-α message, or specific controllers of theTNF-α convertases could be present in different cell types. It is alsonot necessary that the TNF-α convertases cleave and process onlyproTNF-α; they could have other substrates as well. It also may be thatdifferent TNF-α convertases process proTNF-α in different cell types.Thus, one convertase might carry out processing in T and NK cells, whilea different enzyme might function in macrophages. It therefore may notbe necessary that a given TNF-α convertase be present in all cell typesthat make TNF-α.

[0028] But apart from the requirement that a TNF-α convertase of thisinvention be present in at least one type of cell that makes TNF-α,whether the other possibilities discussed in the foregoing paragraph arecorrect or not is not essential to the invention.

[0029] Tryptic digestion followed by amino acid sequencing of a peptidefrom a TNF-α convertase isolated from bovine spleen revealed that enzymeto be further characterized by an amino acid sequence comprising asequence substantially as follows:

[0030] Met-Asn-Ser-Leu-Leu-Gly/Asp-Ser-Ala-Pro (SEQ ID NO: 2).

[0031] This bovine enzyme is further characterized by behavior observedin various chromatographic systems during purification, as is describedin detail in the Example below, and by an apparent molecular weight inSDS-PAGE under reducing conditions of about 65 kDa. The presentinvention also encompasses another bovine TNF-α convertase and enzymesfrom other mammalian species, including human TNF-α convertases.

[0032] Some general properties of TNF-α convertases as defined in thisinvention are as follows:

[0033] (1) Inhibited by ethylenediaminetetraacetic acid (EDTA),dithiothreitol (DTT), 1,10-phenanthroline and/or α-2 macroglobulin.

[0034] (2) Not inhibited by serine, cysteine and acid proteaseinhibitors, such as 100 μM captopril, 300 μM phosphoramidon, 100 μMthiorphan, 100 μM dichloroisocoumarin (DCI), 1 mM iodoacetic acid (IAA),1 μg/ml tissue inhibitor of metalloproteases-1 (TIMP-1), 1 μg/ml soybeantrypsin inhibitor (SBTI), 1 mMmethoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (AAPV) and 100 μMtrans-epoxysuccinyl-L-leucylamino (4-guanidino)-butane (E64; a thiolprotease inhibitor).

[0035] (3) Membrane-bound on THP-1 cells and on other monocytic-typecells and cell lines.

[0036] (4) Cleave human pro-TNF-α at an Ala-Val peptide bond, to producesoluble, mature TNF-α.

[0037] The proteins of the present invention are useful in rational drugdiscovery screens for the identification of compounds that selectivelyblock the conversion of proTNF-α to soluble, mature TNF-α. They havethis utility because when introduced into cells used in the screens,e.g., by transfection of nucleic acids encoding the proteins, theyproduce TNF-α convertase activity which can act on an appropriatesubstrate as described herein. Inhibitors can be identified by measuringinhibition of this activity.

[0038] Some Definitions

[0039] As used herein, the term “bovine TNF-α convertase” in oneembodiment means an enzyme having the above-mentioned subsequence andpurification characteristics, or a significant fragment of such aprotein which substantially retains the proteolytic activity andspecificity disclosed herein. In another embodiment, “bovine TNF-αconvertase” means bovine ADAM 10 [GenBank Accession No. Z21961;Wolfsberg et al., J. Cell. Biol. 131:275 (1995)], which the presentinventors have surprisingly found is a TNF-α convertase. It also refersto a bovine-derived enzyme exhibiting similar enzymatic activity whichspecifically binds to an antibody elicited against either of the bovineTNF-α convertases, or to a proteolytically active fragment from one ofthose enzymes. Such antibodies typically bind to a bovine or other TNF-αconvertase with high affinity, e.g., with an affinity constant of atleast about 100 nM, usually better than about 30 nM, preferably betterthan about 10 nM, and more preferably better than about 3 nM.

[0040] Because bovine ADAM 10 is a TNF-α convertase and a comparison ofits amino acid sequence with available sequence information on humanADAM 10 shows the two proteins to be 96% homologous, the presentinventors believe that human ADAM 10 is also a TNF-α convertase asdefined herein.

[0041] Surprisingly, the present inventors have also discovered that thehuman membrane-type metalloproteases MT-MMP1 [Sato et al., Nature 370:61(1994)], MT-MMP2 [Will et al., Eur. J.

[0042] Biochem. 231:602 (1995)] and MT-MMP3 [Takino et al., J. Biol.Chem. 270:23013 (1995)] are also TNF-α convertases as defined herein.

[0043] The present inventors have further cloned a cDNA encoding a novelhuman protein. When transfected into mammalian cells otherwise incapableof processing human proTNF-α to soluble, mature TNF-α, this proteinproduces such processing. The sequence of this DNA, together with thepredicted amino acid sequence, is substantially as defined in theSequence Listing by SEQ ID NO: 21.

[0044] As used herein, the term “polypeptide” means a fragment orsegment, e.g., of a TNF-α convertase which comprises a subsequence ofthe complete amino acid sequence of the enzyme containing at least about8, preferably at least about 12, more preferably at least about 20, andmost preferably at least about 30 or more contiguous amino acidresidues, up to and including the total number of residues in thecomplete enzyme.

[0045] The polypeptides of the invention can comprise any part of thecomplete sequence of a TNF-α convertase. Thus, although they could beproduced by proteolytic cleavage of an intact enzyme, they can also bemade by chemical synthesis or by the application of recombinant DNAtechnology and are not limited to polypeptides delineated by proteolyticcleavage sites.

[0046] The term “analog(s)” means a TNF-α convertase which has beenmodified by deletion, addition, modification or substitution of one ormore amino acid residues in the wild-type enzyme. It encompasses allelicand polymorphic variants, and also muteins and fusion proteins whichcomprise all or a significant part of a TNF-α convertase, e.g.,covalently linked via a side-chain group or terminal residue to adifferent protein, polypeptide or moiety (fusion partner).

[0047] Some amino acid substitutions are preferably “conservative”, withresidues replaced with physicochemically similar residues, such asGly/Ala, Asp/Glu, Val/Ile/Leu, Lys/Arg, Asn/Gln and Phe/Trp/Tyr. Analogshaving such conservative substitutions typically retain substantialproteolytic activity. Other analogs, which have non-conservativesubstitutions such as Asn/Glu, Val/Tyr and His/Glu, may substantiallylack proteolytic activity. Nevertheless, such analogs are useful becausethey can be used as antigens to elicit production of antibodies in animmunologically competent host. Because these analogs retain many of theepitopes (antigenic determinants) of the wild-type enzymes from whichthey are derived, many antibodies produced against them can also bind tothe active-conformation or denatured wild-type enzymes. Accordingly, theantibodies can be used, e.g., for the immunopurification or immunoassayof the wild-type enzymes.

[0048] Whether a particular analog exhibits convertase activity can bedetermined by routine experimentation as described herein.

[0049] Some analogs are truncated variants in which residues have beensuccessively deleted from the amino- and/or carboxyl-termini, whilesubstantially retaining the characteristic proteolytic activity.

[0050] Modifications of amino acid residues may include but are notlimited to aliphatic esters or amides of the carboxyl terminus or ofresidues containing carboxyl side chains, O-acyl derivatives of hydroxylgroup-containing residues, and N-acyl derivatives of the amino-terminalamino acid or amino-group containing residues, e.g., lysine or arginine.

[0051] This invention also encompasses physical variants havingsubstantial amino acid sequence homology with the amino acid sequencesof the TNF-α convertases or polypeptides. In this invention, amino acidsequence homology, or sequence identity, is determined by optimizingresidue matches and, if necessary, by introducing gaps as required.Homologous amino acid sequences are typically intended to includenatural allelic, polymorphic and interspecies variations in eachrespective sequence.

[0052] Typical homologous proteins or peptides will have from 25-100%homology (if gaps can be introduced) to 50-100% homology (ifconservative substitutions are included), with the amino acid sequenceof the TNF-α convertases. Primate species convertases are of particluarinterest.

[0053] Observed homologies will typically be at least about 35%,preferably at least about 50%, more preferably at least about 75%, andmost preferably at least about 85% or more. See Needleham et al., J.Mol. Biol. 48:443-453 (1970); Sankoff et al. in Time Warps, StringEdits, and Macromolecules: The Theory and Practice of SequenceComparison, 1983, Addison-Wesley, Reading, Mass.; and software packagesfrom IntelliGenetics, Mountain View, Calif., and the University ofWisconsin Genetics Computer Group, Madison, Wis.

[0054] Glycosylation variants include, e.g., analogs made by modifyingglycosylation patterns during synthesis and processing in variousalternative eukaryotic host expression systems, or during furtherprocessing steps. Particularly preferred methods for producingglycosylation modifications include exposing the TNF-α convertases toglycosylating enzymes derived from cells which normally carry out suchprocessing, such as mammalian glycosylation enzymes. Alternatively,deglycosylation enzymes can be used to remove carbohydrates attachedduring production in eukaryotic expression systems.

[0055] Other analogs are TNF-α convertases containing modifications,such as incorporation of unnatural amino acid residues, orphosphorylated amino acid residues such as phosphotyrosine,phosphoserine or phosphothreonine residues. Other potentialmodifications include sulfonation, biotinylation, or the addition ofother moieties, particularly those which have molecular shapes similarto phosphate groups.

[0056] Analogs of TNF-α convertases can be prepared by chemicalsynthesis or by using site-directed mutagenesis [Gillman et al., Gene8:81 (1979); Roberts et al., Nature 328:731 (1987) or Innis (Ed.), 1990,PCR Protocols: A Guide to Methods and Applications, Academic Press, NewYork, N.Y.] or the polymerase chain reaction method [PCR;

[0057] Saiki et al., Science 239:487 (1988)], as exemplified byDaugherty et al. [Nucleic Acids Res. 19:2471 (1991)] to modify nucleicacids encoding the complete enzymes. Adding epitope tags forpurification or detection of recombinant products is envisioned.

[0058] General techniques for nucleic acid manipulation and expressionare described generally, e.g., in Sambrook, et al., Molecular Cloning: ALaboratory Manual (2d ed.), 1989, Vols. 1-3, Cold Spring HarborLaboratory. Techniques for the synthesis of polypeptides are described,for example, in Merrifield, J. Amer. Chem. Soc. 85:2149 (1963);Merrifield, Science 232:341 (1986); and Atherton et al., Solid PhasePeptide Synthesis: A Practical Approach, 1989, IRL Press, Oxford.

[0059] Still other analogs are prepared by the use of agents known inthe art for their usefulness in cross-linking proteins through reactiveside groups. Preferred derivatization sites with cross-linking agentsare free amino groups, carbohydrate moieties and cysteine residues.

[0060] Substantial retention of proteolytic activity by the foregoinganalogs of the TNF-α convertases typically entails retention of at leastabout 50%, preferably at least about 75%, more preferably at least about80%, and most preferably at least about 90% of the proTNF-α processingactivity and/or specificity of the corresponding wild-type enzyme.

[0061] As used herein, the term “isolated nucleic acid” means a nucleicacid such as an RNA or DNA molecule, or a mixed polymer, which issubstantially separated from other components that are normally found incells or in recombinant DNA expression systems. These components includebut are not limited to ribosomes, polymerases, serum components, andflanking genomic sequences. The term thus embraces a nucleic acid whichhas been removed from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analogs oranalogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule.

[0062] An isolated nucleic acid will generally be a homogeneouscomposition of molecules but may, in some embodiments, contain minorheterogeneity. Such heterogeneity is typically found at the ends ofnucleic acid coding sequences or in regions not critical to a desiredbiological function or activity.

[0063] A “recombinant nucleic acid” is defined either by its method ofproduction or structure. Some recombinant nucleic acids are thus made bythe use of recombinant DNA techniques which involve human intervention,either in manipulation or selection. Others are made by fusing twofragments not naturally contiguous to each other. Engineered vectors areencompassed, as well as nucleic acids comprising sequences derived usingany synthetic oligonucleotide process.

[0064] For example, a wild-type codon may be replaced with a redundantcodon encoding the same amino acid residue or a conservativesubstitution, while at the same time introducing or removing a nucleicacid sequence recognition site. Similarly, nucleic acid segmentsencoding desired functions may be fused to generate a single geneticentity encoding a desired combination of functions not found together innature. Although restriction enzyme recognition sites are often thetarget of such artificial manipulations, other site-specific targets,e.g., promoters, DNA replication sites, regulation sequences, controlsequences, or other useful features may be incorporated by design.Sequences encoding epitope tags for detection or purification asdescribed above may also be incorporated.

[0065] A nucleic acid “fragment” is defined herein as a nucleotidesequence comprising at least about 17, generally at least about 25,preferably at least about 35, more preferably at least about 45, andmost preferably at least about 55 or more contiguous nucleotides.

[0066] This invention further encompasses recombinant DNA molecules andfragments having sequences that are identical or highly homologous tothose described herein. The nucleic acids of the invention may beoperably linked to DNA segments which control transcription,translation, and DNA replication.

[0067] “Homologous nucleic acid sequences” are those which when alignedand compared exhibit significant similarities. Standards for homology innucleic acids are either measures for homology generally used in the artby sequence comparison or based upon hybridization conditions, which aredescribed in greater detail below.

[0068] Substantial nucleotide sequence homology is observed when thereis identity in nucleotide residues in two sequences (or in theircomplementary strands) when optimally aligned to account for nucleotideinsertions or deletions, in at least about 50%, preferably in at leastabout 75%, more preferably in at least about 90%, and most preferably inat least about 95% of the aligned nucleotides.

[0069] Substantial homology also exists when one sequence will hybridizeunder selective hybridization conditions to another. Typically,selective hybridization will occur when there is at least about 55%homology over a stretch of at least about 30 nucleotides, preferably atleast about 65% over a stretch of at least about 25 nucleotides, morepreferably at least about 75%, and most preferably at least about 90%over about 20 nucleotides. See, e.g., Kanehisa, Nucleic Acids Res.12:203 (1984).

[0070] The lengths of such homology comparisons may encompass longerstretches and in certain embodiments may cover a sequence of at leastabout 17, preferably at least about 25, more preferably at least about50, and most preferably at least about 75 nucleotide residues.

[0071] Stringency of conditions employed in hybridizations to establishhomology are dependent upon factors such as salt concentration,temperature, the presence of organic solvents, and other parameters.Stringent temperature conditions usually include temperatures in excessof about 30° C., often in excess of about 37° C., typically in excess ofabout 45° C., preferably in excess of about 55° C., more preferably inexcess of about 65° C., and most preferably in excess of about 70° C.Stringent salt conditions will ordinarily be less than about 1000 mM,usually less than about 500 mM, more usually less than about 400 mM,preferably less than about 300 mM, more preferably less than about 200mM, and most preferably less than about 150 mM. For example, saltconcentrations of 100, 50 and 20 mM are used. The combination of theforegoing parameters, however, is more important than the measure of anysingle parameter. See, e.g., Wetmur et al., J. Mol. Biol. 31:349 (1968).

[0072] The term “substantially pure” is defined herein to mean a TNF-αconvertase or other material that is free from other contaminatingproteins, nucleic acids, and other biologicals derived from an originalsource organism or recombinant DNA expression system. Purity may beassayed by standard methods and will typically exceed at least about50%, preferably at least about 75%, more preferably at least about 90%,and most preferably at least about 95% purity. Purity evaluation may bemade on a mass or molar basis.

[0073] Antibody Production

[0074] Antigenic (i.e., immunogenic) fragments of the TNF-α convertasesof this invention, which may or may not have enzymatic activity, maysimilarly be produced. Regardless of whether they cleave proTNF-α, suchfragments, like the complete TNF-α convertases, are useful as antigensfor preparing antibodies, using standard methods, that can bind to thecomplete enzymes. Shorter fragments can be concatenated or attached to acarrier. Because it is well known in the art that epitopes generallycontain at least about five, preferably at least about 8, amino acidresidues [Ohno et al., Proc. Natl. Acad. Sci. USA 82:2945 (1985)],fragments used for the production of antibodies will generally be atleast that size. Preferably, they will contain even more residues, asdescribed above. Whether a given fragment is immunogenic can readily bedetermined by routine experimentation.

[0075] Although it is generally not necessary when complete TNF-αconvertases are used as antigens to elicit antibody production in animmunologically competent host, smaller antigenic fragments arepreferably first rendered more immunogenic by cross-linking orconcatenation, or by coupling to an immunogenic carrier molecule (i.e.,a macromolecule having the property of independently eliciting animmunological response in a host animal). Cross-linking or conjugationto a carrier molecule may be required because small polypeptidefragments sometimes act as haptens (molecules which are capable ofspecifically binding to an antibody but incapable of eliciting antibodyproduction, i.e., they are not immunogenic). Conjugation of suchfragments to an immunogenic carrier molecule renders them moreimmunogenic through what is commonly known as the “carrier effect”.

[0076] Suitable carrier molecules include, e.g., proteins and natural orsynthetic polymeric compounds such as polypeptides, polysaccharides,lipopolysaccharides etc. Protein carrier molecules are especiallypreferred, including but not limited to keyhole limpet hemocyanin andmammalian serum proteins such as human or bovine gammaglobulin, human,bovine or rabbit serum albumin, or methylated or other derivatives ofsuch proteins. Other protein carriers will be apparent to those skilledin the art. Preferably, but not necessarily, the protein carrier will beforeign to the host animal in which antibodies against the fragments areto be elicited.

[0077] Covalent coupling to the carrier molecule can be achieved usingmethods well known in the art, the exact choice of which will bedictated by the nature of the carrier molecule used. When theimmunogenic carrier molecule is a protein, the fragments of theinvention can be coupled, e.g., using water soluble carbodiimides suchas dicyclohexylcarbodiimide or glutaraldehyde.

[0078] Coupling agents such as these can also be used to cross-link thefragments to themselves without the use of a separate carrier molecule.Such cross-linking into aggregates can also increase immunogenicity.Immunogenicity can also be increased by the use of known adjuvants,alone or in combination with coupling or aggregation.

[0079] Suitable adjuvants for the vaccination of animals include but arenot limited to Adjuvant 65 (containing peanut oil, mannide monooleateand aluminum monostearate); Freund's complete or incomplete adjuvant;mineral gels such as aluminum hydroxide, aluminum phosphate and alum;surfactants such as hexadecylamine, octadecylamine, lysolecithin,dimethyldioctadecylammonium bromide,N,N-dioctadecyl-N′,N′-bis(2-hydroxymethyl) propanediamine,methoxyhexadecylglycerol and pluronic polyols; polyanions such as pyran,dextran sulfate, poly IC, polyacrylic acid and carbopol; peptides suchas muramyl dipeptide, dimethylglycine and tuftsin; and oil emulsions.The polypeptides could also be administered following incorporation intoliposomes or other microcarriers.

[0080] Information concerning adjuvants and various aspects ofimmunoassays are disclosed, e.g., in the series by P. Tijssen, Practiceand Theory of Enzyme Immunoassays, 3rd Edition, 1987, Elsevier, NewYork. Other useful references covering methods for preparing polyclonalantisera include Microbiology, 1969, Hoeber Medical Division, Harper andRow; Landsteiner, Specificity of Serological Reactions, 1962, DoverPublications, New York, and Williams, et al., Methods in Immunology andImmunochemistry, Vol. 1, 1967, Academic Press, New York.

[0081] Serum produced from animals immunized using standard methods canbe used directly, or the IgG fraction can be separated from the serumusing standard methods such as plasmaphoresis or adsorptionchromatography with IgG-specific adsorbents such as immobilized ProteinA. Alternatively, monoclonal antibodies can be prepared.

[0082] Hybridomas producing monoclonal antibodies against the TNF-αconvertases of the invention or antigenic fragments thereof are producedby well-known techniques. Usually, the process involves the fusion of animmortalizing cell line with a B-lymphocyte that produces the desiredantibody. Alternatively, non-fusion techniques for generating immortalantibody-producing cell lines can be used, e.g., virally-inducedtransformation [Casali et al., Science 234:476 (1986)]. Immortalizingcell lines are usually transformed mammalian cells, particularly myelomacells of rodent, bovine, and human origin. Most frequently, rat or mousemyeloma cell lines are employed as a matter of convenience andavailability.

[0083] Techniques for obtaining antibody-producing lymphocytes frommammals injected with antigens are well known. Generally, peripheralblood lymphocytes (PBLs) are used if cells of human origin are employed,or spleen or lymph node cells are used from non-human mammalian sources.A host animal is injected with repeated dosages of the purified antigen(human cells are sensitized in vitro), and the animal is permitted togenerate the desired antibody-producing cells before they are harvestedfor fusion with the immortalizing cell line. Techniques for fusion arealso well known in the art, and in general involve mixing the cells witha fusing agent, such as polyethylene glycol.

[0084] Hybridomas are selected by standard procedures, such as HAT(hypoxanthine-aminopterin-thymidine) selection. Those secreting thedesired antibody are selected using standard immunoassays, such asWestern blotting, ELISA (enzyme-linked immunosorbent assay), RIA(radioimmunoassay), or the like. Antibodies are recovered from themedium using standard protein purification techniques [Tijssen, Practiceand Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985)].

[0085] Many references are available to provide guidance in applying theabove techniques [Kohler et al., Hybridoma Techniques (Cold SpringHarbor Laboratory, New York, 1980); Tijssen, Practice and Theory ofEnzyme Immunoassays (Elsevier, Amsterdam, 1985); Campbell, MonoclonalAntibody Technology (Elsevier, Amsterdam, 1984); Hurrell, MonoclonalHybridoma Antibodies: Techniques and Applications (CRC Press, BocaRaton, Fla., 1982)]. Monoclonal antibodies can also be produced usingwell known phage library systems. See, e.g., Huse, et al., Science246:1275 (1989); Ward, et al., Nature 341:544 (1989).

[0086] Antibodies thus produced, whether polyclonal or monoclonal, canbe used, e.g., in an immobilized form bound to a solid support by wellknown methods, to purify the TNF-α convertases by immunoaffinitychromatography.

[0087] Antibodies against the antigenic fragments can also be used,unlabeled or labeled by standard methods, as the basis for immunoassaysof the TNF-α convertases. The particular label used will depend upon thetype of immunoassay. Examples of labels that can be used include but arenot limited to radiolabels such as ³²P, ¹²⁵I, ³H and ¹⁴C; fluorescentlabels such as fluorescein and its derivatives, rhodamine and itsderivatives, dansyl and umbelliferone; chemiluminescers such asluciferia and 2,3-dihydro-phthalazinediones; and enzymes such ashorseradish peroxidase, alkaline phosphatase, lysozyme andglucose-6-phosphate dehydrogenase.

[0088] The antibodies can be tagged with such labels by known methods.For example, coupling agents such as aldehydes, carbodiimides,dimaleimide, imidates, succinimides, bisdiazotized benzadine and thelike may be used to tag the antibodies with fluorescent,chemiluminescent or enzyme labels. The general methods involved are wellknown in the art and are described, e.g., in Immunoassay: A PracticalGuide, 1987, Chan (Ed.), Academic Press, Inc., Orlando, Fla. Suchimmunoassays could be carried out, for example, on fractions obtainedduring purification of the TNF-α convertases.

[0089] The antibodies of the present invention can also be used toidentify particular cDNA clones expressing the TNF-α convertases, inexpression cloning systems.

[0090] Neutralizing antibodies that bind to the catalytic site of aTNF-α convertase may also be used as inhibitors to block substratebinding, and hence catalytic activity. This can be done using completeantibody molecules, or well known antigen binding fragments such as Fab,Fc, F(ab)₂, and Fv fragments.

[0091] Definitions of such fragments can be found, e.g., in Klein,Immunology (John Wiley, New York, 1982); Parham, Chapter 14, in Weir,ed. Immunochemistry, 4th Ed. (Blackwell Scientific Publishers, Oxford,1986). The use and generation of antibody fragments has also beendescribed, e.g.: Fab fragments [Tijssen, Practice and Theory of EnzymeImmunoassays (Elsevier, Amsterdam, 1985)], Fv fragments [Hochman et al.,Biochemistry 12:1130 (1973); Sharon et al., Biochemistry 15:1591 (1976);Ehrlich et al., U.S. Pat. No. 4,355,023] and antibody half molecules(Auditore-Hargreaves, U.S. Pat. No. 4,470,925). Methods for makingrecombinant Fv fragments based on known antibody heavy and light chainvariable region sequences have further been described, e.g., by Moore etal. (U.S. Pat. No. 4,642,334) and by Plückthun [Bio/Technology 9:545(1991)]. Alternatively, they can be chemically synthesized by standardmethods.

[0092] The antibodies and antigen-binding fragments thereof can be usedtherapeutically to block the activity of a TNF-α convertase, and therebyto treat any medical condition caused or mediated by TNF-α. Suchantibodies and fragments are preferably chimeric or humanized, to reduceantigenicity and human anti-mouse antibody (HAMA) reactions. Themethodology involved is disclosed, e.g., in U.S. Pat. No. 4,816,397 toBoss et al. and in U.S. Pat. No. 4,816,567 to Cabilly et al. Furtherrefinements on antibody humanization are described in European Patent451 216 B1.

[0093] The dosage regimen involved in a therapeutic application will bedetermined by the attending physician, considering various factors whichmay modify the action of the antibodies or binding fragments, e.g., thecondition, body weight, sex and diet of the patient, the severity of anyinfection, time of administration, and other clinical factors.

[0094] Typical protocols for the therapeutic administration ofantibodies are well known in the art and have been disclosed, e.g., byElliott et al. [The Lancet 344:1125 (1994)], Isaacs et al. [The Lancet340:748 (1992)], Anasetti et al. [Transplantation 54:844 (1992)],Anasetti et al. [Blood 84:1320 (1994)], Hale et al. [The Lancet 2:1394(Dec. 17, 1988)], Queen [Scrip 1881:18 (1993)] and Mathieson et al. [N.Eng. J. Med. 323:250 (1990)].

[0095] Administration of the compositions of this invention is typicallyparenteral, by intraperitoneal, intravenous, subcutaneous, orintramuscular injection, or by infusion or by any other acceptablesystemic method. Administration by intravenous infusion, typically overa time course of about 1 to 5 hours, is preferred.

[0096] Often, treatment dosages are titrated upward from a low level tooptimize safety and efficacy. Generally, daily antibody dosages willfall within a range of about 0.01 to 20 mg protein per kilogram of bodyweight. Typically, the dosage range will be from about 0.1 to 5 mgprotein per kilogram of body weight.

[0097] Dosages of antigen binding fragments from the antibodies will beadjusted to account for the smaller molecular sizes and possiblydecreased half-lives (clearance times) following administration. Variousmodifications or derivatives of the antibodies or fragments, such asaddition of polyethylene glycol chains (PEGylation), may be made toinfluence their pharmacokinetic and/or pharmacodynamic properties.

[0098] It will be appreciated by those skilled in the art, however, thatthe TNF-α convertase inhibitors of the invention are not limited toneutralizing antibodies or binding fragments thereof. This inventionalso encompasses other types of inhibitors, including small organicmolecules and inhibitory substrate analogs.

[0099] One example of a class of small organic molecule inhibitorspotentially useful in this invention is a metalloprotease inhibitordesignated GI 129471, which has been shown to block TNF-α secretion,both in vitro and in vivo [McGeehan et al., Nature 370:558 (1994)].Another such example isN-{D,L-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl}L-3-(2′naphthyl)-alanyl-Lalanine, 2-aminoethyl amide [Mohler et al., Nature 370:218 (1994)],which has been shown to protect mice against a lethal dose of endotoxin.Still another example of a small organic molecule inhibitor is acompound, designated SCH 43534, which is mentioned in an example below.This compound is a peptide-based hydroxamate inhibitor of collagenasestructurally similar to the foregoing compounds, the inhibitory activityof which validates use of an assay of the invention to identify a TNF-αconvertase inhibitor.

[0100] The foregoing small organic molecules are not specific inhibitorsof a TNF-α convertase but inhibit other metalloproteases as well. As isdescribed more fully below, specific inhibitors of a TNF-α convertasewhich do not inhibit other metalloproteases can also be identified usingthe methods of this invention if desired.

[0101] An “effective amount” of a composition of the invention is anamount that will ameliorate one or more of the well known parametersthat characterize medical conditions caused or mediated by TNF-α. Manysuch parameters and conditions have been described, e.g., as in a reviewby K. J. Tracey in The Cytokine Handbook, Second Edition, A. Thompson,Ed., 1994, Academic Press Ltd., London, UK, pp. 289-304. The referencescited by Tracey are also incorporated herein in their entirety byreference.

[0102] Although the compositions of this invention could be administeredin simple solution, they are more typically used in combination withother materials such as carriers, preferably pharmaceutical carriers.Useful pharmaceutical carriers can be any compatible, non-toxicsubstance suitable for delivering the compositions of the invention to apatient. Sterile water, alcohol, fats, waxes, and inert solids may beincluded in a carrier. Pharmaceutically acceptable adjuvants (bufferingagents, dispersing agents) may also be incorporated into thepharmaceutical composition. Generally, compositions useful forparenteral administration of such drugs are well known; e.g. Remington'sPharmaceutical Science, 17th Ed. (Mack Publishing Company, Easton, Pa.,1990). Alternatively, compositions of the invention may be introducedinto a patient's body by implantable drug delivery systems [Urquhart etal., Ann. Rev. Pharmacol. Toxicol. 24:199 (1984)].

[0103] Therapeutic formulations may be administered in many conventionaldosage formulation. Formulations typically comprise at least one activeingredient, together with one or more pharmaceutically acceptablecarriers. Formulations may include those suitable for oral, rectal,nasal, or parenteral (including subcutaneous, intramuscular, intravenousand intradermal) administration.

[0104] The formulations may conveniently be presented in unit dosageform and may be prepared by any methods well known in the art ofpharmacy. See, e.g., Gilman et al. (eds.) (1990), The PharmacologicalBases of Therapeutics, 8th Ed., Pergamon Press; and Remington'sPharmaceutical Sciences, supra, Easton, Pa.; Avis et al. (eds.) (1993)Pharmaceutical Dosage Forms: Parenteral Medications Dekker, New York;Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: TabletsDekker, New York; and Lieberman et al. (eds.) (1990), PharmaceuticalDosage Forms: Disperse Systems Dekker, New York.

[0105] The present invention also encompasses anti-idiotypic antibodies,both polyclonal and monoclonal, which are produced using theabove-described antibodies as antigens. These antibodies are usefulbecause they may mimic the structures of the proteases.

[0106] Protein Purification

[0107] The proteins, polypeptides and antigenic fragments of thisinvention can be purified by standard methods, including but not limitedto salt or alcohol precipitation, preparative disc-gel electrophoresis,isoelectric focusing, high pressure liquid chromatography (HPLC),reversed-phase HPLC, gel filtration, cation and anion exchange andpartition chromatography, and countercurrent distribution. Suchpurification methods are well known in the art and are disclosed, e.g.,in Guide to Protein Purification, Methods in Enzymology, Vol. 182, M.Deutscher, Ed., 1990, Academic Press, New York, N.Y. More specificmethods applicable to purification of the bovine TNF-α convertases aredescribed below.

[0108] Purification steps can be followed by carrying out assays forTNF-α convertase activity as described below. Particularly where aconvertase is being isolated from a cellular or tissue source, it ispreferable to include one or more inhibitors of other proteolyticenzymes is the assay system. Such inhibitors include, e.g.,4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF),pepstatin, leupeptin, andmethoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (AAPV).

[0109] Nucleic Acids and Expression Systems

[0110] Nucleic acids encoding the TNF-α convertases or fragments thereofcan be prepared by standard methods. For example, DNA can be chemicallysynthesized using, e.g., the phosphoramidite solid support method ofMatteucci et al. [J. Am. Chem. Soc. 103:3185 (1981)], the method of Yooet al. [J. Biol. Chem. 764:17078 (1989)], or other well known methods.This can be done by sequentially linking a series of oligonucleotidecassettes comprising pairs of synthetic oligonucleotides, as describedbelow.

[0111] Of course, due to the degeneracy of the genetic code, manydifferent nucleotide sequences can encode the TNF-α convertases. Thecodons can be selected for optimal expression in prokaryotic oreukaryotic systems. Such degenerate variants are of course alsoencompassed by this invention.

[0112] Moreover, nucleic acids encoding the TNF-α convertases canreadily be modified by nucleotide substitutions, nucleotide deletions,nucleotide insertions, and inversions of nucleotide stretches. Suchmodifications result in novel DNA sequences which encode antigens havingimmunogenic or antigenic activity in common with the wild-type enzymes.These modified sequences can be used to produce wild-type or mutantenzymes, or to enhance expression in a recombinant DNA system.

[0113] Insertion of the DNAs encoding the TNF-α convertases into avector is easily accomplished when the termini of both the DNAs and thevector comprise compatible restriction sites. If this cannot be done, itmay be necessary to modify the termini of the DNAs and/or vector bydigesting back single-stranded DNA overhangs generated by restrictionendonuclease cleavage to produce blunt ends, or to achieve the sameresult by filling in the single-stranded termini with an appropriate DNApolymerase.

[0114] Alternatively, desired sites may be produced, e.g., by ligatingnucleotide sequences (linkers) onto the termini. Such linkers maycomprise specific oligonucleotide sequences that define desiredrestriction sites. Restriction sites can also be generated by the use ofthe polymerase chain reaction (PCR). See, e.g., Saiki et al., Science239:487 (1988). The cleaved vector and the DNA fragments may also bemodified if required by homopolymeric tailing.

[0115] Expression of nucleic acids encoding the TNF-α convertases ofthis invention can be carried out by conventional methods in eitherprokaryotic or eukaryotic cells. Although strains of E. coli areemployed most frequently in prokaryotic systems, many other bacteriasuch as various strains of Pseudomonas and Bacillus are know in the artand can be used as well.

[0116] Prokaryotic expression control sequences typically used includepromoters, including those derived from the β-lactamase and lactosepromoter systems [Chang et al., Nature 198:1056 (1977)], the tryptophan(trp) promoter system [Goeddel et al., Nucleic Acids Res. 8:4057(1980)], the lambda P_(L) promoter system [Shimatake et al., Nature292:128 (1981)] and the tac promoter [De Boer et al., Proc. Natl. Acad.Sci. USA 292:128 (1983)]. Numerous expression vectors containing suchcontrol sequences are known in the art and available commercially.

[0117] Eukaryotic expression systems typically insect, mammalian oryeast host cells, for which many expression vectors are known in the artand commercially available.

[0118] Screening Systems and Methods

[0119] To identify inhibitors of the TNF-α convertases, the enzymes areemployed in basic screening systems. Essentially, these systems providemethods for bringing together a mammalian TNF-α convertase, anappropriate substrate for the enzyme, and a sample to be tested for thepresence of an inhibitor of the enzyme. If the sample contains such aninhibitor, substantially reduced cleavage of the substrate will beobserved, compared to what would be observed in the absence of aninhibitor, e.g., using a “control” sample containing only buffer.

[0120] A basic screening method comprises:

[0121] (a) contacting a mammalian TNF-α convertase in the presence ofsubstrate with a sample to be tested for the presence of an inhibitor ofthe convertase; and

[0122] (b) measuring the rate of cleavage of the substrate;

[0123] whereby an inhibitor of the TNF-α convertase in the sample isidentified by measuring substantially reduced cleavage of the substrate,compared to what would be measured in the absence of such inhibitor.

[0124] “Substantially reduced cleavage” of a substrate by a TNF-αconvertase inhibitor will be observed by measuring less than about 50%,preferably less than about 25%, more preferably less than about 10%, andmost preferably less than about 5% of the cleavage measured in theabsence of an inhibitor.

[0125] The term “sample” is defined herein to mean any solution, whetheraqueous, organic of some combination of the two, that may contain aTNF-α convertase inhibitor. Examples of samples include but are notlimited to solutions of compounds obtained following organic synthesis,aliquots from purification step fractions, and extracts from cells ortissues, or from other biological or microbial materials.

[0126] TNF-α convertase substrates that can be used in the basic assaysof the invention include polypeptides comprising the complete proTNF-αsequence, and truncated variants (polypeptides) thereof, the preferredrequirement being that all substrates contain the specific Ala-Val bond,the cleavage of which characterizes the TNF-α convertases of theinvention. Some examples of such substrates are described below,including a protein (SEQ ID NO: 3) and a polypeptide (SEQ ID NO: 4)substrate. Others are known in the art, such as those disclosed byMohler et al. [Nature 370:218 (1995)]. The term “substrate” is definedherein to mean all such materials. Substrates suitable for use in theassays are preferably based on human proTNF-α, although it may bepossible to use substrates from other species.

[0127] The substrates can be engineered so that the activity of a TNF-αconvertase causes a positive or negative measurable change in thesubstrate. This may result in a loss or gain of a measurable signal,following cleavage of the substrate.

[0128] Any TNF-α convertase can be used in the basic screening methodsof this invention, although use of a primate or human enzyme ispreferred for the identification of compounds suitable for use as humantherapeutics. In connection with the assays, the term “TNF-α convertase”encompasses both the wild-type variants and analogs, such as truncatedor substituted variants, as long as they possess substantial proteolyticactivity as defined herein. Use of a wild-type, full-length human enzymeis however preferred. Whether a given analog would be suitable for usein an assay of the invention can readily be determined through routineexperimentation, using the disclosed methods.

[0129] Those skilled in the art will appreciate that there are many waysa mammalian TNF-α convertase could be brought together with a substrateand a test sample to identify an inhibitor, and all such methods arewithin the scope of this invention. Nevertheless, in a preferredembodiment, a mammalian cell system is employed in which one or morenucleic acids encoding a mammalian TNF-α convertase and a substrate aretransfected into a host cell. These nucleic acids can be contained in asingle recombinant vector or in two, as is the case in an Example below.

[0130] Particularly preferred mammalian host cells for use in theforegoing system inherently lack or have minimal ability to cleaveproTNF-α to the mature, secreted form. Examples of such a cell are the293 human embryonic kidney cell line and clones derived therefrom. The293 line is available from the American Type Culture Collection,Rockville, Md., under Accession No. ATCC CRL 1573. A clone derived fromthe 293 line, designated 293EBNA, is available from Invitrogen.

[0131] TNF-α convertase inhibitors identified in the basic screens ofthis invention may be suitable for therapeutic administration, althoughthey may also inhibit other metalloproteases. If it is desired toidentify a specific inhibitor of a TNF-α convertase, i.e., one that willnot inhibit the activity of other, more general metalloproteases, thatcan be done using another embodiment of the present invention.

[0132] As used herein, the term “specific inhibitor of a TNF-αconvertase” is defined to mean an inhibitor which blocks the proteolyticactivity of a TNF-α convertase but does not inhibit the activity ofcollagenase or other matrix-degrading metalloproteases.

[0133] The following is a summary of some matrix-degradingmetalloproteases, including some names by which they have been calledand some of their substrates: Enzyme Names Substrates InterstitialCollagenase (MMP-1) Collagens I, II, III, VII and X 72-kDa Gelatinase(MMP-2) Collagens IV, V, VII and X Stromelysin (MMP-3) Collagens III,IV, V and IX Uterine Metalloproteinase Gelatins I, III, IV and V (MMP-7)Neutrophil Collagenase (MMP-8) Collagens I, II and III 92-kDa Gelatinase(MMP-9) Collagens IV and V Stromelysin-2 (MMP-10) Gelatins I, III, IVand V

[0134] More information on the names and substrates, and on theproperties of the above-mentioned matrix-degrading metalloproteases, canbe found in a review by Woessner [FASEB J. 5:2145 (1991)].

[0135] To identify a specific inhibitor of a TNF-α convertase, thescreening methods of the invention further comprise:

[0136] (a) contacting a matrix-degrading metalloprotease in the presenceof substrate with an inhibitor of a TNF-α convertase; and

[0137] (b) measuring the rate of cleavage of the substrate;

[0138] whereby a specific inhibitor of a TNF-α convertase is identifiedby measuring substantially undiminished cleavage of the substrate,compared to what would be measured in the absence of such inhibitor.

[0139] In a preferred embodiment, a mammalian cell system is employed inwhich one or more nucleic acids encoding a matrix-degradingmetalloprotease and a substrate are transfected into a host cell. Thesenucleic acids can be contained in a single recombinant vector or in two.

[0140] “Substantially undiminished cleavage” of a substrate by aspecific inhibitor of a TNF-α convertase will be observed by measuringat least about 75%, preferably at least about 90%, more preferably atleast about 95%, and most preferably at least about 99% of the cleavagemeasured in the absence of such an inhibitor.

[0141] Molecular Cloning and Expression

[0142] The present invention provides methods for cloning bovine TNF-αconvertase and corresponding enzymes from other mammalian species.Briefly, Southern and Northern blot analysis can be carried out toidentify cells from other species expressing genes encoding the TNF-αconvertases. Complementary DNA (cDNA) libraries can be prepared bystandard methods from mRNA isolated from such cells, and degenerateprobes or PCR primers based on the amino acid sequence informationprovided herein can be used to identify clones encoding a TNF-αconvertase.

[0143] Alternatively, expression cloning methodology can be used toidentify particular clones encoding a TNF-α convertase. An antibodypreparation which exhibits cross-reactivity with TNF-α convertases froma number of mammalian species may be useful in monitoring expressioncloning.

[0144] Preferably, a co-transfection system described more fully belowis used to identify clones capable of cleaving proTNF-α to the mature,secreted form. Selected clones can then be amplified, and cDNA isolatedfrom them can be inserted into vectors suitable for expression inprokaryotic or eukaryotic expression systems.

[0145] Briefly, this method for identifying a nucleic acid encoding amammalian TNF-α convertase comprises:

[0146] (a) culturing a mammalian host cell comprising a firstrecombinant expression vector comprising a nucleic acid encoding a TNF-αconvertase substrate and a second recombinant expression vectorcomprising a nucleic acid that is to be tested to determine whether itencodes a mammalian TNF-α convertase, under conditions in whichexpression occurs; and

[0147] (b) measuring the rate of cleavage of the substrate;

[0148] whereby a nucleic acid encoding a mammalian TNF-α convertase isidentified by measuring substantially increased cleavage of thesubstrate, compared to what would be measured in the absence of suchnucleic acid.

[0149] Preferably the TNF-α convertase substrate used is proTNF-α,although any of the other substrates mentioned herein could be usedinstead.

[0150] In the context of this invention, “substantially increasedcleavage” of the substrate will be observed by measuring at least about5 times more, preferably at least about 10 times more, more preferablyat least about 25 times more, and most preferably at least about 50times more cleavage of the substrate than would occur in the absence ofa nucleic acid encoding a mammalian TNF-α convertase.

[0151] However identified, clones encoding TNF-α convertases fromvarious mammalian species can be isolated and sequenced, and the codingregions can be excised and inserted into an appropriate vector.

[0152] Recombinant expression vectors in this invention are typicallyself-replicating DNA or RNA constructs comprising nucleic acids encodingone of the TNF-α convertases, usually operably linked to suitablegenetic control elements that are capable of regulating expression ofthe nucleic acids in compatible host cells. Genetic control elements mayinclude a prokaryotic promoter system or a eukaryotic promoterexpression control system, and typically include a transcriptionalpromoter, an optional operator to control the onset of transcription,transcription enhancers to elevate the level of mRNA expression, asequence that encodes a suitable ribosome binding site, and sequencesthat terminate transcription and translation. Expression vectors alsomay contain an origin of replication that allows the vector to replicateindependently of the host cell.

[0153] Vectors that could be used in this invention include microbialplasmids, viruses, bacteriophage, integratable DNA fragments, and othervehicles which may facilitate integration of the nucleic acids into thegenome of the host. Plasmids are the most commonly used form of vectorbut all other forms of vectors which serve an equivalent function andwhich are, or become, known in the art are suitable for use herein. See,e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985 andSupplements, Elsevier, N.Y., and Rodriguez et al. (eds.), Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, 1988, Buttersworth,Boston, Mass.

[0154] Suitable host cells for expressing nucleic acids encoding theTNF-α convertases include prokaryotes and higher eukaryotes. Prokaryotesinclude both gram negative and positive organisms, e.g., E. coli and B.subtilis. Higher eukaryotes include established tissue culture celllines from animal cells, both of non-mammalian origin, e.g., insectcells, and birds, and of mammalian origin, e.g., human, primates, androdents.

[0155] Prokaryotic host-vector systems include a wide variety of vectorsfor many different species. As used herein, E. coli and its vectors willbe used generically to include equivalent vectors used in otherprokaryotes. A representative vector for amplifying DNA is pBR322 ormany of its derivatives. Vectors that can be used to express the TNF-α.convertases include but are not limited to those containing the lacpromoter (pUC-series); trp promoter (pBR322-trp); Ipp promoter (thepIN-series); lambda-pP or pR promoters (pOTS); or hybrid promoters suchas ptac (pDR540). See Brosius et al., “Expression Vectors EmployingLambda-, trp-, lac-, and Ipp-derived Promoters”, in Rodriguez andDenhardt (eds.) Vectors: A Survey of Molecular Cloning Vectors and TheirUses, 1988, Buttersworth, Boston, pp. 205-236.

[0156] Higher eukaryotic tissue culture cells are preferred hosts forthe recombinant production of enzymatically active TNF-α convertases.Although any higher eukaryotic tissue culture cell line might be used,including insect baculovirus expression systems, mammalian cells arepreferred. Transformation or transfection and propagation of such cellshas become a routine procedure. Examples of useful cell lines includeHeLa cells, Chinese hamster ovary (CHO) cell lines, baby rat kidney(BRK) cell lines, insect cell lines, bird cell lines, and monkey (COS)cell lines. Expression vectors for such cell lines usually include anorigin of replication, a promoter, a translation initiation site, RNAsplice sites (if genomic DNA is used), a polyadenylation site, and atranscription termination site. These vectors also usually contain aselection gene or amplification gene. Suitable expression vectors may beplasmids, viruses, or retroviruses carrying promoters derived, e.g.,from such sources as adenovirus, SV40, parvoviruses, vaccinia virus, orcytomegalovirus. Representative examples of suitable expression vectorsinclude pCDNA1, pCD [Okayama et al., Mol. Cell Biol. 5:1136 (1985)],pMC1neo Poly-A [Thomas et al., Cell 51:503 (1987)], pUC19, pREP8,pSVSPORT and derivatives thereof, and baculovirus vectors such as pAC373 or pAC 610.

EXAMPLES

[0157] The present invention can be illustrated by the followingexamples.

[0158] Unless otherwise indicated, percentages given below for solids insolid mixtures, liquids in liquids, and solids in liquids are on awt/wt, vol/vol and wt/vol basis, respectively. Sterile conditions weregenerally maintained during cell culture.

[0159] General Methods

[0160] Standard methods were used, as described, e.g., in Maniatis etal., Molecular Cloning: A Laboratory Manual, 1982, Cold Spring HarborLaboratory, Cold Spring Harbor Press; Sambrook et al., MolecularCloning: A Laboratory Manual, (2d ed.), Vols 1-3, 1989, Cold SpringHarbor Press, NY; Ausubel et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements), CurrentProtocols in Molecular Biology, Greene/Wiley, New York; Innis et al.(eds.) PCR Protocols: A Guide to Methods and Applications, 1990,Academic Press, N.Y.

[0161] In vitro Assays for TNF-α Convertase Activity

[0162] For the assay of convertase activity associated with or isolatedfrom membrane preparations, a protein-based assay was carried outessentially as described by Mohler et al., Nature 370:218 (1995).

[0163] Briefly, a peptide-tagged recombinant human TNF-α proteinsubstrate (Flag-TNF-α) was cloned and expressed in E. coli. The proteinwas purified by affinity chromatography using an M2(anti-Flag)-Sepharose column (Kodak), and by ion exchange chromatographyusing a BIOCAD equipped with an HQ10 column.

[0164] The amino acid sequence of the protein substrate is defined inthe Sequence Listing by SEQ ID NO: 3, concerning which the followingshould be noted. Amino acid residues 2-9 comprise the “Flag” sequence.Residues 10 and 11 are a Gly-Ser connector following the Flag which wereadded to accommodate a restriction site used in construction. Thehistidine at position 12 corresponds to the histidine at position −25 ofSEQ ID NO: 1, after which the two sequences are identical to thecarboxyl termini. Thus, fifty residues of the normal leader sequence ofhuman proTNF-α have been deleted from this substrate. Since the deletedregion is not essential for use as a substrate in an assay of thisinvention, other truncations containing deletion of more or fewerresidues could be used as well.

[0165] To test for convertase activity, a membrane protein sample (12μg) was mixed (in 12 μl) with 2 ng of ¹²⁵I-polypeptide substrate(approximately 50,000 cpm) in the presence of inhibitors of otherproteolytic enzymes [e.g., 200 μM 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride (AEBSF), 2 μM pepstatin, 200 μM leupeptin, 1 mMmethoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (AAPV)], and with orwithout 2.5 mM EDTA. Following incubation at 37° C. overnight, sampleswere fractionated in a 16% sodium dodecylsulfate (SDS) gel and subjectedto SDS polyacrylamide gel electrophoresis [SDS-PAGE; Laemmli, Nature227:680 (1970)]. The gels were dried, and radioactivity was detected byautoradiography.

[0166] Examination of the gels revealed intensified bands from thesamples incubated without EDTA, at the position expected for the 17 kDahuman TNF-α cleavage product.

[0167] A polypeptide-based assay for TNF-α convertase activity was alsocarried out essentially using the method of Mohler et al., Supra.Briefly, an amino-terminal dinitrophenylated peptide substrate, amidatedat the carboxyl terminus (SEQ ID NO: 4) was synthesized and purifiedusing standard procedures. Dinitrophenylated polypeptides correspondingto cleavage products expected for TNF-α convertase (a dinitrophenylatedpeptide having the sequence of residues 1-6 of SEQ ID NO: 4) and forother membrane proteases were also synthesized.

[0168] To test for convertase activity, a membrane protein sample wasmixed in 50 μl with 1 μg peptide substrate in the presence of inhibitors(0.2 mM AEBSF, 2 μM pepstatin, 200 μM leupeptin, 1 mM AAPV). Followingincubation at 37° C. for from 60 minutes to overnight, the protein wasprecipitated by cold 5% trichloroacetic acid (TCA), 20% acetonitrile,and the soluble peptide fraction was applied to a YMC 120 angstrom C-18ODS-AQ column (4.6×100 mm; YMC, Inc.). The column was elutedisocratically at a flow rate of 1 ml/min using 40% acetonitrile plus0.06 % trifloroacetic acid. The elution of DNP-peptide was monitored at360 nm using a Waters 625 LC system.

[0169] The results of a typical assay are shown in FIG. 1, wherein thepositions of the uncut peptide and an expected convertase cleavageproduct (DNP-APLAQA) are shown. Of course, other cleavage productsattributable to the activities of other proteases present in the crudemembrane sample were also observed; some of their elution positions arealso shown in FIG. 1.

[0170] Preparation and Sequencing of Bovine TNF-α Convertases

[0171] Bovine spleen purchased from Pel-Freeze was cut into smallpieces, washed in cold PBS, and then shredded using a Black & DeckerPOWER PRO food processor. The tissue was resuspended in lysis buffer [20mM Tricine (N-[2-Hydroxy-1,1-bis(hydroxymethyl)-ethyl] glycine), pH 7.8,8% sucrose] containing 0.1 % PMSF, then homogenized using a BrinkmannPOLYTRON tissue homogenizer. Cell debris was removed by twocentrifugations at 8,000' g, and the membranes were isolated byultracentrifugation at 60,000× g. The isolated membranes were washed in10 mM Hepes, pH 7.5, then resuspended and frozen at −20° C. until used.

[0172] The membrane fraction was thawed and resuspended to aconcentration of 8 mg/ml protein in Buffer A (20 mM Tris, pH 7.5, 1 mMMgSO₄, 10 mM NaCl, 10 μM ZnSO₄) plus 2% Brij 35 (23 lauryl ether), andincubated at 4° C. for 30 minutes. The membranes were collected byultracentrifugation at 60,000× g, then resuspended to 4 mg/ml in bufferA plus 2% Lubrol (polyethylene glycol monododecyl ether). The insolubleprotein was removed by ultracentrifugation at 60,000× g. TheBrij-insoluble, lubrol-solubilized membrane protein fraction wasadjusted to 0.3 M NaCl and applied to a chelating Sepharose columncharged with nickel sulfate. The column was washed and eluted with washbuffer (Buffer A with 0.3 M NaCl and 0.1% octyl glucoside) plus 50 mMimidazole. The eluate was concentrated by ultrafiltration, then appliedto a S300 sieving column equilibrated in buffer A plus 0.1%octylglucoside. A retained fraction (corresponding to a molecular weightof approximately 60,000) was pooled, adjusted to 0.3 M NaCl and appliedto a wheat germ agglutinin column.

[0173] The retained fraction was eluted with 0.5 M N-acetyl glucosaminein Buffer A with 0.5 M NaCl, dialyzed against 1 mM sodium phosphatebuffer, pH 7, and applied to a hydroxyapatite column. The unboundfraction was passed over an HQ-10 ion exchange column (PerseptiveBiosystems), and eluted with a linear gradient of from 0 to 500 mM NaCl.Fractions containing TNF-α convertase activity were pooled for furthercharacterization.

[0174] The final protein fraction (approximately 10 jig) of the firstbovine TNF-α convertase was applied to an 8% polyacrylamide gel inSDS-glycine buffer (Novex). Following electrophoresis, the gel wasstained for 8 minutes in 10% acetic acid/50% methanol containing 0.1 %Coomassie blue, then destained for 3.5 hours in three changes of 10%acetic acid/50% methanol. The polypeptide was excised and subjected toin situ tryptic cleavage, peptide isolation and microsequencing usingstandard methods.

[0175] This enzyme was a bovine TNF-α convertase, amino acid sequencesof which are disclosed above.

[0176] The purification of bovine ADAM 10 was carried out by subjectingbovine spleen to the procedures described above to the point ofapplication to the wheat germ agglutinin column. Thereafter, theretained fraction was eluted with 0.5 M N-acetyl glucosamine, dialyzedagainst 1 mM sodium phosphate buffer, pH 7, and applied to anhydroxyapatite column. A bound fraction from that column was passed overan HQ-10 ion exchange column.

[0177] The unbound fraction, containing the TNF-α convertase activity(approximately 10 μg) was subjected to electrophoresis an 8%polyacrylamide gel using SDS-glycine buffer (Novex) with dithiothreitol.Following electrophoresis, the fractionated proteins wereelectophoretically transferred to an IMMOBILON filter membrane in 10 mMCAPS (3-[Cyclohexylamino]-1-propanesulfonic acid) buffer, pH 10, with10% methanol. The protein band was visualized by staining the membrane0.1 % Ponceau S in 40% methanol, 10% acetic acid, excised, and subjectedto in situ tryptic cleavage, peptide isolation and microsequencing,including N-terminal analysis, using standard methods.

[0178] Cloning of Human MT-MMP1, MT-MMP2, MT-MMP3, MMP7, MMP12 andBovine ADAM 10

[0179] Human MT-MMP1 cDNA was cloned from THP-1 cell (ATCC TIB 202)total RNA, which was converted to single-stranded DNA using a GibcoBRLSUPERSCRIPT Preamplification System (Catalog #18089-011) and an oligo dTprimer. This DNA was then used directly for PCR using primers designated#5261 (SEQ ID NO: 5; 5′ forward primer) and #5271 (SEQ ID NO: 6; 3′reverse primer). PCR conditions were: 94° C., 30 seconds/60° C.,seconds/72° C., 2 minutes, for 30 cycles. The PCR product was cut withKpnI/HindIII and ligated into similarly cut pSVSPORT (GibcoBRL).

[0180] Cloning of human MT-MMP2 was initiated by isolating total RNAfrom THP-1 cells and preparing single-stranded DNA as described above.The DNA was then subjected to a two-step PCR protocol to obtain thefull-length MT-MMP2 cDNA as follows.

[0181] First, two PCR reactions were run that encompassed overlappingfront and back halves of MT-MMP2. One reaction was set up using primersdesignated #B5295GD (SEQ ID NO: 7; 5′ forward) and “reverse internal”(SEQ ID NO: 8; internal 3′). A second PCR reaction was set up usingprimers #B5296GD (SEQ ID NO: 9; 3′ reverse) and “forward internal” (SEQID NO: 10; internal 5′). PCR conditions were as described above. Theproduct of each of these reactions was isolated by agarose gelelectrophoresis.

[0182] In the second step, the two products from the PCR reactions weremixed with PCR primers B5295GD and B5296GD, and PCR was performed underthe same conditions as described above. The product from this reactionwas cut with EcoRI/XbaI, isolated by agarose gel electrophoresis, andcloned into vector pSRαSPORT that had been cut with the same restrictionenzymes.

[0183] Human MT-MMP3 was cloned as described for MT-MMP1 but from aortapolyA⁺ RNA (Clontech) using PCR primers designated #5322 (SEQ ID NO: 11;5′ forward primer) and #5323 (SEQ ID NO: 12; 3′ reverse primer). The PCRproduct was cut with KpnI/HindIII, isolated, and cloned into vectorpSRαSPORT that had been cut with the same restriction enzymes.

[0184] Similarly, human MMP7 (matrilysin) was cloned from human testispoly A⁺ RNA (Clontech) and using PCR primers #5367 (SEQ ID NO: 13; 5′forward primer) and #5369 (SEQ ID NO: 14; 3′ reverse primer). The PCRproduct was cut with KpnI/HindIII, isolated, and cloned into vectorpSRαSPORT that had been cut with the same restriction enzymes.

[0185] Human MMP12 (macrophage metalloelastase) was similarly clonedfrom human aorta polyA⁺ RNA using PCR primers #A0698H03 (SEQ ID NO: 15;5′ forward primer) and #A0698H08 (SEQ ID NO: 16; 3′ reverse primer). ThePCR product was cut with KpnI/XbaI and, following isolation, cloned intosimilarly-cut vector pSRαSPORT.

[0186] Bovine ADAM 10 was cloned as described above from 5 μg of totalRNA isolated from bovine spleen poly A⁺ RNA (Clontech). The resultingsingle-stranded DNA was then used for PCR, using primers havingsequences defined in the Sequence listing by SEQ ID NO: 17 (5′ forwardprimer) and SEQ ID NO: 18 (3′ reverse primer). PCR conditions were asdescribed above, and the PCR product was digested with KpnI and HindIIIand ligated into similarly-digested vector pCEP4 (Invitrogen).

[0187] Construction of a Human proTNF-α Expression Vector

[0188] Human proTNF-α cDNA can be cloned from total RNA isolated fromLPS (lipopolysaccharide)-stimulated THP-1 cells and converted tosingle-stranded DNA as described above. This DNA can then be used forPCR, e.g., using primers designed to introduce BamHI cleavage sites intothe PCR product. The sequences of suitable 5′ and 3′ primers are definedin the Sequence Listing by SEQ ID NO: 19 and SEQ ID NO: 20,respectively. The PCR product is then cut with BamHI to produce aninsert that can be cloned into a BamHI-cleaved expression vector.

[0189] A similar insert was ligated into pUC19 (New England Biolabs)that had been cut with BamHI, to produce a vector designated pUCTNF.Vector pUCTNF was then cut with SalI/HindIII, and a fragment retainingthe coding region for proTNF-α was ligated into a vector designatedpSRαSPORT that had cut with the same restriction enzymes. VectorpSRαSPORT had previously been constructed as follows.

[0190] pSVSPORT 1 (GibcoBRL) was cut with ClaI/PstI to remove the SVpromoter and treated with Klenow polymerase to fill in the ClaI overhangand produce a blunt end. A fragment containing the SRα promoter and SV40t antigen was obtained following cleavage of plasmid pDSRG (ATCC 68233;International Patent Application Publication No. WO 91/01078) withHindIII/PstI and filling of the HindIII overhang with Klenow polymerase.This fragment was then ligated into the cut pSVSPORT 1 to producepSRαSPORT.

[0191] Cloning of a Novel Human Protein

[0192] Vector pSRαSPORT was cleaved using NotI/SalI, and a 1.5 kbstuffer cDNA fragment was ligated into the cut vector. The stuffer cDNAfragment, which was a neomycin resistance gene, was prepared bydigesting plasmid PMC1neo Poly A (Stratagene, Catalog No. 213201) withXhoI/SalI, and the small fragment was isolated and ligated intoSalI-digested pSL1190 (Pharmacia, catalog No. 27-4386). The stufferfragment was released by digesting the resulting vector pSC1190-Neo withSalI, and the 1.5 kb fragment was isolated.

[0193] The construct incorporating the stuffer fragment was cleavedusing NotI/SalI, and the linearized vector was separated from the insertcDNA by agarose gel electrophoresis. The cleaved vector was thenrepurified in a second agarose electrophoresis gel. The pure cleavedvector DNA was isolated from the agarose gel using GELZYME (Invitrogen),following the recommended conditions. This vector was used to clonelibrary cDNA.

[0194] Messenger RNA was prepared by treating Mono Mac-6 cells[Ziegler-Heitbrock et al., Int. J. Cancer 41:456 (1988)] withlipopolysaccharide at 1 μg/ml for 18 hours, and with PMA at 10 ng/ml forone hour prior to RNA isolation. Total RNA was prepared from these cellsusing the guanidine thiocyanate method (Sambrook et al., supra, pp.7.19-7.22). The cells were collected by centrifugation, resuspended inguanidine thiocyanate (Gibco BRL) with 2.5 grams of N-laurylsarcosinesodium salt (Sigma), 5 drops of anti-foam A (Sigma) and 0.75 ml of2-mercaptoethanol (Biorad).

[0195] The lysate was layered over an equal volume of 5.7 M cesiumchloride solution and centrifuged in an SW41 rotor for 24 hours at25,000 rpm. The resulting total RNA pellet was washed with ethanol,resuspended in sterile water, and treated with DNAse by incubating 500μg of total RNA per ml in buffer containing 5 units of RQ1 DNAse I(Promega), 400 units of RNAsin (Promega), 10 mM MgCl₂, and 5 mM DTT at37° C. for 30 min. The solution was treated with an equal volume of 1:1phenol/chloroform solution, and the RNA was precipitated with ethanol.PolyA⁺ mRNA was isolated from the total RNA using the OLIGOTEX mRNAisolation system (Qiagen Inc.).

[0196] Five micrograms of mRNA were used to synthesize cDNA followingthe protocols in the SUPERSCRIPT Plasmid System for cDNA synthesis andplasmid cloning (Gibco BRL), with the following modifications. Followingsecond strand synthesis, the cDNA was phenol/chloroform extracted,ethanol precipitated, and then treated with T4 DNA Polymerase (PharmaciaLKB), following the manufacturer's instructions. Following NotIdigestion, the resuspended cDNA was subjected to electrophoresis in 1%SEAPLAQUE GTG agarose (FMC) and visualized by ethidium bromide staining.The portion of the gel from 2 kb to 13 kb was excised and digested withGELZYME (Invitrogen).

[0197] The resulting size-enriched cDNA was ligated with theNotI/SalI-cleaved pSRαSport vector overnight using a 2:1 vector/insertconcentration ratio. The ligation mixture was then extracted withphenol/chloroform, precipitated with ethanol, and electroporated intoELECTROMAX DH10B cells (Gibco BRL) under the prescribed conditions. Thecells were plated out at a density of about 1000 colonies per plate,with a total of around 7×10⁵ colonies for the entire library.

[0198] Cells from each plate were collected in 1.5 ml Luria broth. Analiquot (500 μl) of each pool was mixed with 250 μl of 80% (v/v)glycerol, and stored at −20° C. The remaining cells were collected bycentrifugation, and plasmid DNA isolated using the QIAWELL 8 UltraPlasmid Kit (Qiagen) following the recommended procedures. Final DNApreparation was eluted from the Qiawell resin with two 100 μl aliquotsof 10 mM Tris pH8.

[0199] Positives from an initial 800 plasmid pools were identified usingthe cell transfection assay described below and then split into pools of50 colonies per plate, and DNA was prepared as described above.Positives from secondary assays were then spread onto Petri plates, and144 individual colonies were picked. DNA was prepared from each clone asdescribed above, and all positive pools were reconfirmed in duplicate byretransfection and ELISA at each step before proceeding.

[0200] Plasmid DNA was sequenced by the Taq DyeDeoxy™ Terminator CycleSequencing kit and method (Perkin-Elmer/Applied Biosystems) in twodirections using an Applied Biosystems Model 373A DNA Sequencer.Sequence alignment was performed using ABI SEQED Analysis and SequenceNavigator software. The results are shown in SEQ ID NO: 21, whichprovides the complete open reading frame nucleotide sequence togetherwith the predicted amino acid sequence.

[0201] When co-transfected into 293EBNA cells with the vector encodinghuman proTNF-α as described below, the novel cDNA caused the productionof soluble, mature TNF-α. This activity was inhibited by SCH 43534.

[0202] Cell Transfection TNF-α Convertase Assay System

[0203] To develop an effective cell-based assay system, it was firstnecessary to identify host cells that substantially lacked TNF-αconvertase activity. This was accomplished by transiently transfecting anumber of cell types with an expression vector encoding a substrate suchas human proTNF-α, and then assaying for the expected cleavage products,as is explained more fully below.

[0204] It thus was established that although COS cells possesssignificant TNF-α convertase activity, the human embryonic kidney cellline 293 and a clone derived therefrom, designated 293EBNA, lacked theability to cleave transfected human proTNF-α. That was true even thoughall of the cells when transfected with a vector expressing a humangrowth hormone as a control secreted that product. For that reason,293/293EBNA cells are preferred transfection hosts for the assaydescribed below.

[0205] This assay system was established using nucleic acids encodingknown membrane-type matrix metalloproteases, some of which possess TNF-αconvertase activity as defined herein. Using this system in conjunctionwith a vector(s) encoding one of the specific human membrane-type matrixmetalloproteases or bovine ADAM 10, and human proTNF-α, specific TNF-αconvertase inhibitors can be identified.

[0206] Moreover, the same system can also be used to identify othernucleic acids encoding other mammalian TNF-α convertases as well. Thatcan be accomplished by substituting nucleic acids from cDNA or otherlibraries for the nucleic acids encoding the exemplary matrixmetalloproteases, and observing TNF-α convertase activity expressedthereby.

[0207] To demonstrate use of this basic assay system, host cells weretransfected in one of two ways. In one method, human 293EBNA cells(Invitrogen) at 5×10⁶/0.25 ml of RPMI with 10% fetal bovine serum (FBS)were placed in a 0.4 cm electroporation-cuvette with 5 μg total DNA. Thecells were electroporated using a GENE PULSER (BioRad) at 200V, 960 μFd,and 100 ohms. After recovering for 5 minutes, the cells were dilutedinto 15 ml of medium and placed in a tissue culture flask at 37° C., 5%CO₂.

[0208] In a second method, transfections were carried out usingLipofectin (GibcoBRL). DNA (2 μg total) was mixed with 10 μl ofLipofectin in 200 μl of serum free medium (Opti-MEM, GibcoBRL) at roomtemperature for 15 minutes. Then 600 μl of Opti-MEM was added and themixture added to 10⁶ 293EBNA cells. After 4 hours at 37° C., 200 μl of50% FBS were added, and the supernatant was collected 24-48 hours laterfor analysis. For smaller numbers of cells the conditions were scaleddown proportionally.

[0209] The Lipofectin method was also used to assay for bovine ADAM 10activity, whereby 1 μl of Lipofectin was diluted into 10 μl of OPTI-MEMmedium and allowed to stand at room temperature for 30 minutes. Thesolution was mixed with 10 μl of OPTI-MEM containing 50 ng ofproTNFα-SRαSPORT with or without 100 ng of ADAM10-pCEP4, and allowed tostand at room temperature for 15 minutes. Sixty μl of OPTI-MEM wereadded, and the entire 80 μl were used to replace the spent growth mediumin the seeded wells. After incubation at 37° C., 5% CO₂ for 5 hours, 20μl of DME containing 50% fetal bovine serum were added to the well.Following incubation at 37° C., 5% CO₂ for 20 hours, the medium wasassayed for TNFα production.

[0210] To detect convertase activity, enzyme-linked immunosorbant assay(ELISA) was carried out by diluting a human TNF-α capture antibody(Pharmingen #18631D) to 1 μg/ml in 0.1 M NaHCO₃, pH 8.2, and coating thesolution onto a 96 well Nunc MAXISORP microtiter plate at 100 μl/wellovernight at 4° C. The wells were then blocked with 200 μl/well of PBScontaining 10% FBS and 0.1% azide and stored at 4° C. until used forassay.

[0211] Immediately prior to use, the microtiter wells were washed withPBS containing 0.05% Tween 20 (polyoxyethylenesorbitan monolaurate).Samples and standards were diluted in PBS containing 10% FBS, added at100 μl/well, and incubated at 37° C. for 2 hours. After washing asdescribed above, 100 μl of biotinylated anti-TNF-α(Pharmingen #18642D)were added at 1 μg/ml in PBS with 10% FBS and incubated for 45 minutesat 22° C. After washing twice with PBS and Tween 20, streptavidin-HRP(BioSource) was added at a 1:50 dilution, 100 μl/well in PBS with 10%FBS, and incubated for 30 minutes at 22° C. After washing three timeswith PBS and Tween 20, ABTS substrate (KP Labs) was added at 100μl/well, and color development was stopped at an appropriate timedetermined by visual inspection by addition of 100 μl/well of 1% SDS.The plates were read at 405 nm using a Molecular Devices microtiterplate reader.

[0212] Cleavage of proTNF-α by Human MT-MMP1, MT-MMP2 and MT-MMP3 and byBovine ADAM 10

[0213] DNA encoding MT-MMP1 or MT-MMP3 described above was cloned intoexpression vector pREP8 (Invitrogen) and co-transfected with the vectorencoding human proTNF-α into 293EBNA cells. Following an assay carriedout as described above, it was observed that both of the matrixmetalloproteases caused cleavage of the proTNF-α. This is evident inFIG. 2, where the results produced using proTNF-α alone (Bar 1),proTNF-α plus MT-MMP1 (Bar 2), and proTNF-α plus MT-MMP3 (Bar 3) areshown. Similar activity was not shown by MMP7 or by MMP12.

[0214] It was further found that as little as 6 pg/transfection ofMT-MMP1 produced a clear secreted TNF-α signal by ELISA. This wasdetermined by varying the amount of MT-MMP1 vector in the presence of aconstant amount of proTNF-α (50 ng/transfection) and empty pREP8 vector(100 ng/transfection). Thus, this system is useful for identifying otherTNF-α convertases by expression cloning.

[0215] Although the data are not shown, similar results were obtainedfor MT-MMP2.

[0216] Similar results were also obtained using bovine ADAM 10.

[0217] TNF-α Convertase Inhibitor Assay

[0218] To demonstrate use of the transfection assay system to detectinhibitors of a human TNF-α convertase, 293EBNA cells wereco-transfected with expression vectors encoding human proTNF-α andMT-MMP1 as described above, and assayed in the presence or absence ofvarying amounts of an MMP inhibitor, designated SCH 43534, which hadpreviously been shown to block the release of TNF-α from activated humanTHP-1 cells.

[0219] The results are shown in FIG. 3 for proTNF-α alone (Bar 1),proTNF-α plus MT-MMP1 (Bar 2), proTNF-α plus MT-MMP1 plus 1 μM SCH 43534(Bar 3), and proTNF-α plus MT-MMP1 plus 10 μM SCH 43534 (Bar 4).

[0220] Similar results were obtained using bovine ADAM 10. It thus isclear that the present assay can be used to detect inhibitors of TNF-αconvertase activity as defined herein.

[0221] Many modifications and variations of this invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, together with the full scope ofequivalents to which such claims are entitled.

1 21 699 base pairs nucleic acid double linear The residue at position-14 of the sequence is a phenylanine in the Swiss-Prot sequence,Accession Code Swiss-Prot P01375, and as a serine in the GenBanksequence, Accession Number M10988. 1 ATG AGC ACT GAA AGC ATG ATC CGG GACGTG GAG CTG GCC GAG GAG GCG 48 Met Ser Thr Glu Ser Met Ile Arg Asp ValGlu Leu Ala Glu Glu Ala -75 -70 -65 CTC CCC AAG AAG ACA GGG GGG CCC CAGGGC TCC AGG CGG TGC TTG TTC 96 Leu Pro Lys Lys Thr Gly Gly Pro Gln GlySer Arg Arg Cys Leu Phe -60 -55 -50 -45 CTC AGC CTC TTC TCC TTC CTG ATCGTG GCA GGC GCC ACC ACG CTC TTC 144 Leu Ser Leu Phe Ser Phe Leu Ile ValAla Gly Ala Thr Thr Leu Phe -40 -35 -30 TGC CTG CTG CAC TTT GGA GTG ATCGGC CCC CAG AGG GAA GAG TTC CCC 192 Cys Leu Leu His Phe Gly Val Ile GlyPro Gln Arg Glu Glu Xaa Pro -25 -20 -15 AGG GAC CTC TCT CTA ATC AGC CCTCTG GCC CAG GCA GTC AGA TCA TCT 240 Arg Asp Leu Ser Leu Ile Ser Pro LeuAla Gln Ala Val Arg Ser Ser -10 -5 1 TCT CGA ACC CCG AGT GAC AAG CCT GTAGCC CAT GTT GTA GCA AAC CCT 288 Ser Arg Thr Pro Ser Asp Lys Pro Val AlaHis Val Val Ala Asn Pro 5 10 15 20 CAA GCT GAG GGG CAG CTC CAG TGG CTGAAC CGC CGG GCC AAT GCC CTC 336 Gln Ala Glu Gly Gln Leu Gln Trp Leu AsnArg Arg Ala Asn Ala Leu 25 30 35 CTG GCC AAT GGC GTG GAG CTG AGA GAT AACCAG CTG GTG GTG CCA TCA 384 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn GlnLeu Val Val Pro Ser 40 45 50 GAG GGC CTG TAC CTC ATC TAC TCC CAG GTC CTCTTC AAG GGC CAA GGC 432 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu PheLys Gly Gln Gly 55 60 65 TGC CCC TCC ACC CAT GTG CTC CTC ACC CAC ACC ATCAGC CGC ATC GCC 480 Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile SerArg Ile Ala 70 75 80 GTC TCC TAC CAG ACC AAG GTC AAC CTC CTC TCT GCC ATCAAG AGC CCC 528 Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile LysSer Pro 85 90 95 100 TGC CAG AGG GAG ACC CCA GAG GGG GCT GAG GCC AAG CCCTGG TAT GAG 576 Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro TrpTyr Glu 105 110 115 CCC ATC TAT CTG GGA GGG GTC TTC CAG CTG GAG AAG GGTGAC CGA CTC 624 Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly AspArg Leu 120 125 130 AGC GCT GAG ATC AAT CGG CCC GAC TAT CTC GAC TTT GCCGAG TCT GGG 672 Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala GluSer Gly 135 140 145 CAG GTC TAC TTT GGG ATC ATT GCC CTG 699 Gln Val TyrPhe Gly Ile Ile Ala Leu 150 155 9 amino acids amino acid linear There isan ambiquity in the residue at position 6 of the sequence, which may beeither a Gly or an Asp. 2 Met Asn Ser Leu Leu Xaa Ser Ala Pro 1 5 193amino acids amino acid linear 3 Met Asp Tyr Lys Asp Asp Asp Asp Lys GlySer His Phe Gly Val Ile 1 5 10 15 Gly Pro Gln Arg Glu Glu Ser Pro ArgAsp Leu Ser Leu Ile Ser Pro 20 25 30 Leu Ala Gln Ala Val Arg Ser Ser SerArg Thr Pro Ser Asp Lys Pro 35 40 45 Val Ala His Val Val Ala Asn Pro GlnAla Glu Gly Gln Leu Gln Trp 50 55 60 Leu Asn Arg Arg Ala Asn Ala Leu LeuAla Asn Gly Val Glu Leu Arg 65 70 75 80 Asp Asn Gln Leu Val Val Pro SerGlu Gly Leu Tyr Leu Ile Tyr Ser 85 90 95 Gln Val Leu Phe Lys Gly Gln GlyCys Pro Ser Thr His Val Leu Leu 100 105 110 Thr His Thr Ile Ser Arg IleAla Val Ser Tyr Gln Thr Lys Val Asn 115 120 125 Leu Leu Ser Ala Ile LysSer Pro Cys Gln Arg Glu Thr Pro Glu Gly 130 135 140 Ala Glu Ala Lys ProTrp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe 145 150 155 160 Gln Leu GluLys Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp 165 170 175 Tyr LeuAsp Phe Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala 180 185 190 Leu12 amino acids amino acid linear The residue at position 1 is Ser thathas been dinitrophenylated at the a-amino group; that at position 12 isArg, the carboxyl terminus of which has been amidated. 4 Xaa Pro Leu AlaGln Ala Val Arg Ser Ser Ser Xaa 1 5 10 31 base pairs nucleic acid singlelinear 5 TAG CGG TAC CGC CCA CAC TGC CCG GCT GAC C 31 34 base pairsnucleic acid single linear 6 CAA CAA GCT TAC CAC CAC CTT GCT GAC ACT GGTC 34 32 base pairs nucleic acid single linear 7 ACT AGA ATT CAG AGC ATGGGC AGC GAC CCG AG 32 22 base pairs nucleic acid single linear 8 GCC CTTGAA CAC GAA CAT CTC C 22 31 base pairs nucleic acid single linear 9 ACTATC TAG AGC CCC CTG AGC ACC GTT AGC A 31 22 base pairs nucleic acidsingle linear 10 GCC ACA GCC TAC CCA GCC TCT C 22 35 base pairs nucleicacid single linear 11 TAG CGG TAC CAC AGT TCA CTA TGA TCT TAC TCA CA 3535 base pairs nucleic acid single linear 12 TAG CAA GCT TAG CCT GCT CCTAGC TAG GAA ACA GC 35 28 base pairs nucleic acid single linear 13 ACTAGG TAC CAT GCG ACT CAC CGT GCT G 28 29 base pairs nucleic acid singlelinear 14 CAC AAG CTT GCT CAC CGC CCC GCC GCC CT 29 32 base pairsnucleic acid single linear 15 CTA CGG TAC CAC AAT GAA GTT TCT TCT AAT AC32 28 base pairs nucleic acid single linear 16 CTT CTC TAG ACT AAC AACCAA ACC AGC T 28 36 base pairs nucleic acid single linear 17 CTT CCG GGTACC CGG AAG ATG GTG TTG CTG AGA GTG 36 36 base pairs nucleic acid singlelinear 18 CGT TAA AAG CTT TTA ACG TCT CAT GTG TCC CAT CTG 36 31 basepairs nucleic acid single linear 19 ACC GGG ATC CAT GAG CAC TGA AAG CATGAT C 31 32 base pairs nucleic acid single linear 20 GTC TGG ATC CGA ATCCCA GGT TTC GAA GTG GT 32 2352 base pairs nucleic acid double linear 21ATG CCA CAT ACT TTG TGG ATG GTG TGG GTC TTG GGG GTC ATC ATC AGC 48 MetPro His Thr Leu Trp Met Val Trp Val Leu Gly Val Ile Ile Ser 1 5 10 15CTC TCC AAG GAA GAA TCC TCC AAT CAG GCT TCT CTG TCT TGT GAC CGC 96 LeuSer Lys Glu Glu Ser Ser Asn Gln Ala Ser Leu Ser Cys Asp Arg 20 25 30 AATGGT ATC TGC AAG GGC AGC TCA GGA TCT TTA AAC TCC ATT CCC TCA 144 Asn GlyIle Cys Lys Gly Ser Ser Gly Ser Leu Asn Ser Ile Pro Ser 35 40 45 GGG CTCACA GAA GCT GTA AAA AGC CTT GAC CTG TCC AAC AAC AGG ATC 192 Gly Leu ThrGlu Ala Val Lys Ser Leu Asp Leu Ser Asn Asn Arg Ile 50 55 60 65 ACC TACATT AGC AAC AGT GAC CTA CAG AGG TGT GTG AAC CTC CAG GCT 240 Thr Tyr IleSer Asn Ser Asp Leu Gln Arg Cys Val Asn Leu Gln Ala 70 75 80 CTG GTG CTGACA TCC AAT GGA ATT AAC ACA ATA GAG GAA GAT TCT TTT 288 Leu Val Leu ThrSer Asn Gly Ile Asn Thr Ile Glu Glu Asp Ser Phe 85 90 95 TCT TCC CTG GGCAGT CTT GAA CAT TTA GAC TTA TCC TAT AAT TAC TTA 336 Ser Ser Leu Gly SerLeu Glu His Leu Asp Leu Ser Tyr Asn Tyr Leu 100 105 110 TCT AAT TTA TCGTCT TCC TGG TTC AAG CCC CTT TCT TCT TTA ACA TTC 384 Ser Asn Leu Ser SerSer Trp Phe Lys Pro Leu Ser Ser Leu Thr Phe 115 120 125 130 TTA AAC TTACTG GGA AAT CCT TAC AAA ACC CTA GGG GAA ACA TCT CTT 432 Leu Asn Leu LeuGly Asn Pro Tyr Lys Thr Leu Gly Glu Thr Ser Leu 135 140 145 TTT TCT CATCTC ACA AAA TTG CAA ATC CTG AGA GTG GGA AAT ATG GAC 480 Phe Ser His LeuThr Lys Leu Gln Ile Leu Arg Val Gly Asn Met Asp 150 155 160 ACC TTC ACTAAG ATT CAA AGA AAA GAT TTT GCT GGA CTT ACC TTC CTT 528 Thr Phe Thr LysIle Gln Arg Lys Asp Phe Ala Gly Leu Thr Phe Leu 165 170 175 GAG GAA CTTGAG ATT GAT GCT TCA GAT CTA CAG AGC TAT GAG CCA AAA 576 Glu Glu Leu GluIle Asp Ala Ser Asp Leu Gln Ser Tyr Glu Pro Lys 180 185 190 AGT TTG AAGTCA ATT CAG AAT GTA AGT CAT CTG ATC CTT CAT ATG AAG 624 Ser Leu Lys SerIle Gln Asn Val Ser His Leu Ile Leu His Met Lys 195 200 205 210 CAG CATATT TTA CTG CTG GAG ATT TTT GTA GAT GTT ACA AGT TCC GTG 672 Gln His IleLeu Leu Leu Glu Ile Phe Val Asp Val Thr Ser Ser Val 215 220 225 GAA TGTTTG GAA CTG CGA GAT ACT GAT TTG GAC ACT TTC CAT TTT TCA 720 Glu Cys LeuGlu Leu Arg Asp Thr Asp Leu Asp Thr Phe His Phe Ser 230 235 240 GAA CTATCC ACT GGT GAA ACA AAT TCA TTG ATT AAA AAG TTT ACA TTT 768 Glu Leu SerThr Gly Glu Thr Asn Ser Leu Ile Lys Lys Phe Thr Phe 245 250 255 AGA AATGTG AAA ATC ACC GAT GAA AGT TTG TTT CAG GTT ATG AAA CTT 816 Arg Asn ValLys Ile Thr Asp Glu Ser Leu Phe Gln Val Met Lys Leu 260 265 270 TTG AATCAG ATT TCT GGA TTG TTA GAA TTA GAG TTT GAT GAC TGT ACC 864 Leu Asn GlnIle Ser Gly Leu Leu Glu Leu Glu Phe Asp Asp Cys Thr 275 280 285 290 CTTAAT GGA GTT GGT AAT TTT AGA GCA TCT GAT AAT GAC AGA GTT ATA 912 Leu AsnGly Val Gly Asn Phe Arg Ala Ser Asp Asn Asp Arg Val Ile 295 300 305 GATCCA GGT AAA GTG GAA ACG TTA ACA ATC CGG AGG CTG CAT ATT CCA 960 Asp ProGly Lys Val Glu Thr Leu Thr Ile Arg Arg Leu His Ile Pro 310 315 320 AGGTTT TAC TTA TTT TAT GAT CTG AGC ACT TTA TAT TCA CTT ACA GAA 1008 Arg PheTyr Leu Phe Tyr Asp Leu Ser Thr Leu Tyr Ser Leu Thr Glu 325 330 335 AGAGTT AAA AGA ATC ACA GTA GAA AAC AGT AAA GTT TTT CTG GTT CCT 1056 Arg ValLys Arg Ile Thr Val Glu Asn Ser Lys Val Phe Leu Val Pro 340 345 350 TGTTTA CTT TCA CAA CAT TTA AAA TCA TTA GAA TAC TTG GAT CTC AGT 1104 Cys LeuLeu Ser Gln His Leu Lys Ser Leu Glu Tyr Leu Asp Leu Ser 355 360 365 370GAA AAT TTG ATG GTT GAA GAA TAC TTG AAA AAT TCA GCC TGT GAG GAT 1152 GluAsn Leu Met Val Glu Glu Tyr Leu Lys Asn Ser Ala Cys Glu Asp 375 380 385GCC TGG CCC TCT CTA CAA ACT TTA ATT TTA AGG CAA AAT CAT TTG GCA 1200 AlaTrp Pro Ser Leu Gln Thr Leu Ile Leu Arg Gln Asn His Leu Ala 390 395 400TCA TTG GAA AAA ACC GGA GAG ACT TTG CTC ACT CTG AAA AAC TTG ACT 1248 SerLeu Glu Lys Thr Gly Glu Thr Leu Leu Thr Leu Lys Asn Leu Thr 405 410 415AAC ATT GAT ATC AGT AAG AAT AGT TTT CAT TCT ATG CCT GAA ACT TGT 1296 AsnIle Asp Ile Ser Lys Asn Ser Phe His Ser Met Pro Glu Thr Cys 420 425 430CAG TGG CCA GAA AAG ATG AAA TAT TTG AAC TTA TCC AGC ACA CGA ATA 1344 GlnTrp Pro Glu Lys Met Lys Tyr Leu Asn Leu Ser Ser Thr Arg Ile 435 440 445450 CAC AGT GTA ACA GGC TGC ATT CCC AAG ACA CTG GAA ATT TTA GAT GTT 1392His Ser Val Thr Gly Cys Ile Pro Lys Thr Leu Glu Ile Leu Asp Val 455 460465 AGC AAC AAC AAT CTC AAT TTA TTT TCT TTG AAT TTG CCG CAA CTC AAA 1440Ser Asn Asn Asn Leu Asn Leu Phe Ser Leu Asn Leu Pro Gln Leu Lys 470 475480 GAA CTT TAT ATT TCC AGA AAT AAG TTG ATG ACT CTA CCA GAT GCC TCC 1488Glu Leu Tyr Ile Ser Arg Asn Lys Leu Met Thr Leu Pro Asp Ala Ser 485 490495 CTC TTA CCC ATG TTA CTA GTA TTG AAA ATC AGT AGG AAT GCA ATA ACT 1536Leu Leu Pro Met Leu Leu Val Leu Lys Ile Ser Arg Asn Ala Ile Thr 500 505510 ACG TTT TCT AAG GAG CAA CTT GAC TCA TTT CAC ACA CTG AAG ACT TTG 1584Thr Phe Ser Lys Glu Gln Leu Asp Ser Phe His Thr Leu Lys Thr Leu 515 520525 530 GAA GCT GGT GGC AAT AAC TTC ATT TGC TCC TGT GAA TTC CTC TCC TTC1632 Glu Ala Gly Gly Asn Asn Phe Ile Cys Ser Cys Glu Phe Leu Ser Phe 535540 545 ACT CAG GAG CAG CAA GCA CTG GCC AAA GTC TTG ATT GAT TGG CCA GCA1680 Thr Gln Glu Gln Gln Ala Leu Ala Lys Val Leu Ile Asp Trp Pro Ala 550555 560 AAT TAC CTG TGT GAC TCT CCA TCC CAT GTG CGT GGC CAG CAG GTT CAG1728 Asn Tyr Leu Cys Asp Ser Pro Ser His Val Arg Gly Gln Gln Val Gln 565570 575 GAT GTC CGC CTC TCG GTG TCG GAA TGT CAC AGG ATA GCA CTG GTG TCT1776 Asp Val Arg Leu Ser Val Ser Glu Cys His Arg Ile Ala Leu Val Ser 580585 590 GGC ATG TGC TGT GCT CTG TTC CTG CTG ATC CTG CTC ACG GGG GTC CTG1824 Gly Met Cys Cys Ala Leu Phe Leu Leu Ile Leu Leu Thr Gly Val Leu 595600 605 610 TGC CAC CGT TTC CAT GGC CTG TGG TAT ATG AAA ATG ATG TGG GCCTGG 1872 Cys His Arg Phe His Gly Leu Trp Tyr Met Lys Met Met Trp Ala Trp615 620 625 CTC CAG GCC AAA AGG AAG CCC AGG AAA GCT CCC AGC AGG AAC ATATGT 1920 Leu Gln Ala Lys Arg Lys Pro Arg Lys Ala Pro Ser Arg Asn Ile Cys630 635 640 TAT GAT GCA TTT GTT TCT TAC AGT GAG CGG GAT GCC TAC TGG GTGGAG 1968 Tyr Asp Ala Phe Val Ser Tyr Ser Glu Arg Asp Ala Tyr Trp Val Glu645 650 655 AAC CTA ATG GTC CAG GAG CTG GAG AAC TTC AAT CCC CCC TTC AAGTTG 2016 Asn Leu Met Val Gln Glu Leu Glu Asn Phe Asn Pro Pro Phe Lys Leu660 665 670 TGT CTT CAT AAG CGG GAC TTC ATT CCT GGC AAG TGG ATC ATT GACAAT 2064 Cys Leu His Lys Arg Asp Phe Ile Pro Gly Lys Trp Ile Ile Asp Asn675 680 685 690 ATC ATT GAC TCC ATT GAA AAG AGC CAC AAA ACT GTC TTT GTGCTT TCT 2112 Ile Ile Asp Ser Ile Glu Lys Ser His Lys Thr Val Phe Val LeuSer 695 700 705 GAA AAC TTT GTG AAG AGT GAG TGG TGC AAG TAT GAA CTG GACTTC TCC 2160 Glu Asn Phe Val Lys Ser Glu Trp Cys Lys Tyr Glu Leu Asp PheSer 710 715 720 CAT TTC CGT CTT TTT GAT GAG AAC AAT GAT GCT GCC ATT CTCATT CTT 2208 His Phe Arg Leu Phe Asp Glu Asn Asn Asp Ala Ala Ile Leu IleLeu 725 730 735 CTG GAG CCC ATT GAG AAA AAA GCC ATT CCC CAG CGC TTC TGCAAG CTG 2256 Leu Glu Pro Ile Glu Lys Lys Ala Ile Pro Gln Arg Phe Cys LysLeu 740 745 750 CGG AAG ATA ATG AAC ACC AAG ACC TAC CTG GAG TGG CCC ATGGAC GAG 2304 Arg Lys Ile Met Asn Thr Lys Thr Tyr Leu Glu Trp Pro Met AspGlu 755 760 765 770 GCT CAG CGG GAA GGA TTT TGG GTA AAT CTG AGA GCT GCGATA AAG TCC 2352 Ala Gln Arg Glu Gly Phe Trp Val Asn Leu Arg Ala Ala IleLys Ser 775 780 785

What is claimed is:
 1. An isolated bovine TNF-α convertase characterizedby: (a) an amino acid sequence comprising a sequence defined by SEQ IDNO: 2, (b) an apparent molecular weight in SDS-PAGE of about 65 kDa, and(c) an ability to cleave human proTNF-α to produce soluble, mature humanTNF-α.
 2. An antibody which specifically binds to the bovine TNF-αconvertase of claim
 1. 3. The antibody of claim 2 which is a monoclonalantibody.
 4. An isolated or recombinant nucleic acid encoding the bovineTNF-α convertase of claim
 1. 5. A recombinant vector comprising thenucleic acid of claim
 4. 6. A host cell comprising the recombinantvector of claim
 5. 7. A method for making a TNF-α convertase comprisingculturing a host cell of claim 6 under conditions in which the nucleicacid is expressed.
 8. The method of claim 7 in which the convertase isisolated from the culture.
 9. A method for identifying an inhibitor of amammalian TNF-α convertase, comprising: (a) contacting a mammalian TNF-αconvertase in the presence of substrate with a sample to be tested forthe presence of an inhibitor of the convertase; and (b) measuring therate of cleavage of the substrate; whereby an inhibitor of the TNF-αconvertase in the sample is identified by measuring substantiallyreduced cleavage of the substrate, compared to what would be measured inthe absence of such inhibitor.
 10. The method of claim 9 which furthercomprises: (a) contacting a matrix-degrading metalloprotease in thepresence of substrate with an inhibitor of a TNF-α convertase; and (b)measuring the rate of cleavage of the substrate; whereby a specificinhibitor of a TNF-α convertase is identified by measuring substantiallyundiminished cleavage of the substrate, compared to what would bemeasured in the absence of such inhibitor.
 11. The method of claim 10 inwhich the matrix-degrading metalloprotease is selected from the groupconsisting of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9 and MMP-10. 12.The method of claim 9 in which said contacting occurs on the surface ofa mammalian host cell comprising one or more nucleic acids encoding amammalian protein which produces TNF-α convertase activity, and asubstrate of the convertase.
 13. The method of claim 10 in which saidcontacting occurs on the surface of a mammalian host cell comprising oneor more nucleic acids encoding a matrix-degrading metalloprotease and asubstrate of the protease.
 14. The method of claim 12 in which the hostcell is from human embryonic kidney cell line 293 or a clone thereof.15. The method of claim 12 in which the substrate is human proTNF-α. 16.The method of claim 12 in which the mammalian protein is selected fromthe group consisting of bovine ADAM 10; human ADAM 10; human matrix-typemetalloproteases MT-MMP1, MT-MMP2 and MT-MMP3; and a protein comprisingan amino acid sequence defined by SEQ ID NO:
 21. 17. A method foridentifying a nucleic acid encoding a mammalian TNF-α convertase,comprising: (a) culturing a mammalian host cell comprising a firstrecombinant expression vector comprising a nucleic acid encoding a TNF-αconvertase substrate and a second recombinant expression vectorcomprising a nucleic acid that is to be tested to determine whether itencodes a mammalian TNF-α convertase, under conditions in whichexpression occurs; and (b) measuring the rate of cleavage of thesubstrate; whereby a nucleic acid encoding a mammalian TNF-α convertaseis identified by measuring substantially increased cleavage of thesubstrate, compared to what would be measured in the absence of suchnucleic acid.
 18. The method of claim 17 in which the host cell is acell from human embryonic kidney cell line 293 or a clone thereof. 19.The method of claim 17 in which the substrate is human proTNF-α.
 20. Anisolated protein comprising an amino acid sequence defined by SEQ ID NO:21.
 21. An isolated or recombinant nucleic acid encoding the protein ofclaim
 20. 22. The nucleic acid of claim 21 which comprises a nucleotidesequence defined by SEQ ID NO:
 21. 23. A recombinant vector comprising anucleic acid of claim
 21. 24. A host cell comprising a recombinantvector of claim
 23. 25. A method for making a protein comprisingculturing a host cell of claim 24 under conditions in which the nucleicacid is expressed.
 26. The method of claim 25 in which the protein isisolated from the culture.